Methods of monitoring angiogenesis and metastasis in three dimensional co-cultures

ABSTRACT

This disclosure relates to fluorescent cell lines and to the use of such cell lines in monitoring cellular activity, such as angiogenesis. This disclosure further relates to the use of such cell lines in a three-dimensional cell culture to monitor angiogenic and metastatic potential of tumor cells and selecting personalized therapeutics for treatment of cancer.

CROSS REFERENCE TO RELATED APPLICATION

This is a continuation of co-pending U.S. application Ser. No.12/802,666, filed Jun. 10, 2010; which is a continuation-in-part of U.S.application Ser. No. 12/060,752, filed Apr. 1, 2008, now abandoned;which claims the benefit of U.S. Provisional Application No. 60/976,732,filed Oct. 1, 2007. The prior applications are incorporated herein byreference in their entirety.

FIELD OF THE DISCLOSURE

This disclosure relates to fluorescent cell lines and to the use of suchcell lines in monitoring cellular activity, such as angiogenesis, aswell as their use in three-dimensional cell cultures, for instance tomonitor angiogenic and metastatic potential of tumor cells. Alsodescribed are methods of using such cells in selecting personalizedtherapeutics.

BACKGROUND

Biological processes occurring in any organism involve the interactionof multiple cell types, biologically relevant factors, and theorganism's environment. Some of the fundamental questions remaining inbiology are related to understanding how the different cells withinorganisms communicate and organize to form an individual. One frequentlystudied system in which multiple cell types function together andinfluence each other is angiogenesis.

Angiogenesis is a biological process of generating new blood vesselsfrom pre-existing blood vessels into a tissue or organ. Angiogenesis hasbeen intensively studied over the past several decades because of itsfundamental importance in tissue development, vascular diseases, andcancer. Under normal physiological conditions, humans or animals undergoangiogenesis only in very specific restricted situations. For example,angiogenesis is normally observed in fetal and embryonal development andformation of the corpus luteum. Post-natal angiogenesis is an importantphysiological function in the ovary, endometrium, placenta, and in woundhealing.

New vessel growth is tightly controlled by many angiogenic regulators(see for example Folkman, J., Nature Med., 1: 27-31, 1995a), and theswitch of the angiogenesis phenotype depends on the net balance betweenup-regulation of angiogenic stimulators and down-regulation ofangiogenic suppressors. Pathological deregulation of angiogenesis is aprominent feature of a number of human diseases, includingatherogenesis, arthritis, psoriasis, corneal neovascularization,diabetic retinopathy, rheumatoid arthritis, and cancer, for exampleduring malignant transformation that facilitates tumor growth andmetastasis.

In cancer, tumors induce angiogenesis by secreting various growthfactors, such as vascular endothelial growth factor (VEGF), and basicfibroblast growth factor (bFGF) among others. Growth factors, such asbFGF and VEGF, can induce capillary growth into the tumor, which isthought to drive tumor expansion by supplying the tumor with nutrientsand/or removing the cellular waste.

Angiogenesis is also an element of metastasis of a tumor. Single cancercells can break away from an established solid tumor, enter the bloodvessel, and be carried to a distant site, where they can implant andbegin the growth of a secondary tumor. It has even been suggested thatthe blood vessels in a solid tumor may in fact be mosaic vessels,comprised of both endothelial cells and tumor cells. Such mosaicityallows for substantial shedding of tumor cells into the vasculature.

Angiogenesis-based anti-tumor therapies typically use natural andsynthetic angiogenesis inhibitors such as angiostatin, endostatin andtumstatin. Recently the Food and Drug Administration (FDA) approved anantibody therapy targeting angiogenesis in colorectal cancer. Thistherapy is based on a monoclonal antibody directed against an isoform ofVEGF and is marketed under the trade name Avastin®. While establishedanti-angiogenesis therapies are promising, the need still exists for thedevelopment of additional modulators of angiogenesis.

SUMMARY OF THE DISCLOSURE

This disclosure relates to an in vitro assay for use in assessing thecellular activity of cell lines. The assay disclosed herein usesdetectable cell lines from an array of different cell lines, such ascell lines of different cell types and/or anatomical origins, such thatthe effects and interdependency of the different cell lines can bemonitored simultaneously, for example in real-time multiplex assays. Insome examples, the assay uses multiple different cell lines thatcontribute to angiogenesis in vivo, such that the angiogenesis processcan be recapitulated in vitro.

In some embodiments, the disclosed assay uses mammalian cell lines thathave been stably transfected with mammalian expression vectors whichinclude nucleic acid sequences encoding proteins that can be detected bylight emitted by the proteins expressed from the expression vectors, forexample a fluorescent protein expressed from the expression vectors.

In some of the disclosed embodiments, the in vitro assay is a multiplexassay method for evaluating cellular activity, in which a culture isprovided that contains one or more different isolated mammalian celllines (such as histologically different cell lines) that stably andconstitutively express fluorescent proteins having different emissionspectra, for example the different fluorescent proteins have differentwavelengths of emission maxima, such that the emission spectra from thedifferent fluorescent proteins is distinguishable. The culture isassessed for cellular activity by quantifying fluorescence or detectinga pattern of fluorescence from fluorescent proteins present in theculture. In some examples, a culture of two different isolated mammaliancell lines (such as histologically different cell lines) that stably andconstitutively express fluorescent proteins having different emissionspectra is provided and the cellular activity of one or both of thefluorescent cell lines present in the culture is assessed by quantifyingfluorescence or detecting a pattern of fluorescence from fluorescentproteins present in the culture. By extension, the cellular activity ofcell lines present in culture of three, four, five or even more isolatedcell lines expressing fluorescent proteins with different emissionspectra can be assessed by quantifying fluorescence or detecting apattern of fluorescence from the fluorescent proteins present in theculture.

In some embodiments, the cellular activity of the cell line(s) presentin the culture is assessed by determining one or more of the growthrate, migration potential, cell death or tubule formation potential ofthe cell lines using the quantified fluorescence or pattern offluorescence from the fluorescent proteins present in the culture. Suchassays can be used to measure cellular activity and interaction withincomplex biological systems. Such measurements of cellular activity caneven be obtained and/or measured in a temporal sequence or in real-timeas they occur.

In some embodiments, the disclosed in vitro assay is used to determinethe effects of an exogenous agent, such as a test agent (for example apotential modulator of angiogenesis, such as a potential inhibitor ofangiogenesis or a potential stimulator of angiogenesis), growth factor,biological sample (such as a patient sample), another cell line (such asone or more fluorescent cell lines) etc. on the cellular activity of afluorescent cell line. Such an assay can be used to screen formodulators of angiogenesis, for example to identify angiogenesisinhibitors useful in the treatment of cancer.

Also disclosed are cell lines have been stably transfected withmammalian expression plasmids that constitutively express differentfluorescent proteins, for example green fluorescent protein and relatedflorescent proteins, such as yellow fluorescent protein, red fluorescentprotein, cyan fluorescent protein and the like. These cell lines areparticularly suited for use in the disclosed methods. Kits forperforming the disclosed assays, which include the disclosed fluorescentcell lines, are also disclosed.

This disclosure also relates to methods for monitoring angiogenic ormetastatic potential of tumor cells comprising preparing athree-dimensional co-culture that is comprised of three layers. Thefirst layer comprises a neutral polysaccharide polymer gel in contactwith the bottom of the culture dish. The second layer is on top of thefirst layer and comprises a solidified gel matrix, endothelial cellsthat are dispersed in the solidified gel matrix; and tumor cellscomprising either a tumor spheroid colony or a sample of a tumor biopsy,and which are also suspended in the solidified gel matrix, and a thirdlayer comprising culture medium. Angiogenic or metastatic potential oftumor cells is monitored by incubating the three-dimensional co-culture;and detecting at least one of endothelial cell proliferation,endothelial cell tubule formation or tumor cell angiotropism of thecells in the second layer. In particular examples of these methods, thefirst, second, or third layer further comprises at least one test agent,which in some embodiments is a known or potential inhibitor ofangiogenesis or metastasis or augmenter of these processes.

Also disclosed herein are methods of selecting a personalizedanti-angiogenic or anti-metastatic treatment for cancer in a subjectcomprising preparing multiple three-dimensional co-cultures, eachco-culture comprising a first layer comprising a neutral polysaccharidepolymer gel in contact with the bottom of a culture dish; a second layeron top of the first layer, comprising: a solidified gel matrix;endothelial cells dispersed in the solidified gel matrix; and tumorcells comprising either a tumor spheroid colony or a sample of a tumorbiopsy, suspended in the solidified gel matrix; and a third layercomprising culture medium, wherein all but one of the co-culturesfurther comprises at least one test agent comprising an anti-angiogenicor anti-metastatic compound in the first, second, or third layers. Apersonalized anti-angiogenic or anti-metastatic treatment for cancer ina subject is selected by incubating the three-dimensional co-cultures;detecting at least one of endothelial cell proliferation, endothelialcell tubule formation or tumor cell angiotropism of the cells in thesecond layer; and selecting the at least one test agent having thegreatest effect on at least one of endothelial cell proliferation,endothelial cell tubule formation or tumor cell angiotropism incomparison to endothelial cell proliferation, endothelial cell tubuleformation or tumor cell angiotropism in the cells of the co-culturewithout the test agent.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1A is a digital image of porcine aortic endothelial (PAE) cellsthat stably express green fluorescent protein (GFP). FIG. 1B is adigital image of PAE cells that stably express yellow fluorescentprotein (YFP). FIG. 1C is a digital image of PAE cells that stablyexpress red fluorescent protein (RFP). FIG. 1D is a digital image of PAEcells that stably express cyan fluorescent protein (CFP).

FIG. 2 as a graph of fluorescence versus cell number showing linearityof fluorescence versus cell number in mono-cultures of PAE endothelialcells that stably express YFP and mono-cultures of human breastadenocarcinoma cell line MCF7 that stably express RFP.

FIG. 3A is a graph of fluorescence versus cell number showing thatprecise gated fluorescence emission and excitation on YFP allowsdiscrimination of YFP expressing cells (PAE) from RFP expressing cells(MCF7) in co-cultures. FIG. 3B is a graph of fluorescence versus cellnumber showing that precise gated fluorescence emission and excitationon RFP allows discrimination of RFP expressing cells (MCF7) from YFPexpressing cells (PAE) in co-cultures.

FIG. 4A is a graph of growth curves for PAE endothelial cells expressingYFP in mono-culture or in co-culture with MCF7 breast cancer cells. FIG.4B is a graph of growth curves for MCF7 breast cancer cells expressingRFP in mono-culture or in co-culture with PAE endothelial cells.

FIG. 5 is a set of digital images of YFP expressing PAE endothelialcells and RFP expressing MCF7 breast cancer cells in mono-culture andco-culture.

FIG. 6 is a bar graph of the migratory potential of serum obtained from11 subjects and a negative control.

FIG. 7A is a set of digital images showing the tubules formed incultures of PAE endothelial cells at various suramin concentrations.FIG. 7B is a graph of the number of long tubules formed by GFPexpressing PAE cells as a function of suramin concentration. FIG. 7C isa graph of the number of short tubules formed by GFP expressing PAEcells as a function of suramin concentration.

FIG. 8A is a graph showing the migratory potential of fluorescentendothelial cells in response to increasing concentrations of sputumobtained from an Idiopatic Pulmonary Fibrosis (IPF) patient. FIG. 8B isa graph showing the difference in migration potential of sputum from 13normal subjects (NV-BAL) and 13 IPF patients (IPF-BAL) (10% fetal bovineserum (FBS) and phosphate buffered saline (PBS) are controls).

FIG. 9 is a graph of the measured fluorescence present in the cellulargrowth media as a function of increasing TritonX concentration.

FIG. 10 is a flow chart showing an exemplary method for high throughputscreening of test agents (such as small molecules) for antiangiogenicactivity using fluorescent endothelial cells. In one example, a primaryscreen of a small molecule library is done using the disclosed growthand tube formation assays. This primary screen identifies bioactivecompounds, some of which could be cytotoxic. A counterscreen using thedisclosed cell viability assay is used identify those compounds withcytotoxic activity. Biologically active compounds which show nocytotoxicity are considered putative antiangiogenic candidates and canmove forward to in vivo studies.

FIG. 11 shows an exemplary procedure for determining antiangiogenicactivity of a test agent in a multiwell format. In this example, assaysare performed in 96-well plates which contain negative controls(column 1) positive controls (column 12) and 80 remaining wellscontaining the small molecules to be tested. In some examples, a qualitycontrol (Z′ score, Zhang et al. J. Biomol. Screen. 4:67-73, 1999) isapplied to every plate. Only plates with Z values between 0.5 and 1 areconsidered.

FIG. 12 shows a montage of micrographs representing an example of one ofthe growth assay plates included in an exemplary high throughput screen.Column 1 contains cell that have not been stimulated with growth factor(no growth) and column 12 shows growth of the endothelial cells uponexposure to a growth factor cocktail. Test wells show different levelsof cell growth (quantification of the fluorescence in each well is donewith a fluorimeter). Wells which contain growth inhibitors are shown inwhite boxes (hits are defined using the SASD: sum of the average squaredinside-cluster distances, Gagarin et al, J. Biomol. Screen 11:1-12,2006).

FIG. 13A is a graph of cell growth (measured as absolute fluorescence)as a function of time for the wells shown in FIG. 12. FIG. 13B is agraph of cell growth (normalized for the growth rate of the positivecontrol) as a function of time for the wells shown in FIG. 12.

FIG. 14 is a schematic representation of the three-dimensional modelsfor the study of the complex interactions between different cell typesin a three-dimensional environment. Because the different cell typesused are labeled with different fluorescent proteins it becomes easierto image in real time the evolution of the model. It becomes alsopossible to sort apart the cells and do gene expression analysis onthem. Also, these models allow for screening of drugs (antiangiogenic,antitumoral, etc) in a more complex in vitro system.

FIG. 15 is a digital image of a screen shot of the main GUI window ofthe AngioApplication™.

FIG. 16 is a digital image of a screen shot of the settings window ofAngioApplication™.

FIG. 17A is a digital image of a screen shot of the AngioApplication™tubule analysis screen showing a sample under fluorescent illumination.FIG. 17B is a digital image of a screen shot of the AngioApplication™tubule analysis screen showing a sample under bright field illumination.

FIG. 18 is a digital image of a screen shot of the AngioApplication™custom open dialog.

FIG. 19 is a digital image of a screen shot of the excel output of theAngioApplication™.

FIG. 20 is a drawing showing several interactive cellular componentsinvolved with tumor associated angiogenesis/lymphangiogenesis.

FIG. 21 shows 2D and 3D co-culture approaches to simulate the in vivoangiogenic interactions between tumor and endothelial cells.

FIG. 22 is a series of photomicrographs of 2D co-cultures of endothelialcells (PAE) and indicated tumor cell lines. PAE cells were grown on topof matrigel layered on top of a layer of the indicated tumor cell line.PAE seeding density was approximately 18,000 cells/well. Cultures wereincubated for six hours.

FIG. 23 is a series of photomicrographs of 2D co-cultures comparing thetube formation response of different endothelial cells (PAE, HMEC orLEC-1) vs. different tumor cells (A549, CRL-1721 or 92-1). Endothelialcells (at approximately 18,000/well) were plated on top of matrigelsolidified on top of a monolayer of indicated tumor cell line.

FIG. 24 is a series of photographs of human/rat xenograft tumors (A549(left), PC-12 (middle), and 92.1 (right)) excised from nude mice. Toprow shows whole tumors and peripheral vascularization. Bottom row showstumors sectioned to display angiogenesis through tumor core.

FIG. 25 is a series of confocal photomicrographs of 3D co-cultures ofHMEC-1 endothelial cells (yellow) and MCF-7 tumor xenografts (dashedblue ellipse). Left and right panels are magnified 10×. Middle panel ismagnified 5×. Endothelial cells (about 21,000/well) were mixed withsingle ringlet of xenograft core biopsy in molten matrigel that wasallowed to solidify on top of agarose-coated 96 well plates.

FIG. 26 is a confocal photomicrograph of a nine-day 3D co-culture of PAEendothelial cells (red) and 92-1 ocular melanoma (blue).

FIG. 27 is a confocal photomicrograph of a nine-day 3D co-culture ofBEC-1 endothelial cells (red) and rat pheochromocytoma PC-12 (blue).

FIG. 28 is a confocal photomicrograph showing peritumoralvascularization in a twelve-day 3D co-culture of PAE endothelial cells(red) and NSCLC A549 (blue).

FIG. 29 shows confocal photomicrographs of twenty-day 3D co-cultures ofHMEC-1 endothelial cells (red) and human tumor cells (blue). Co-cultureswith ocular melanoma 92.1 (left) and lung cancer A549 (right) are shown.

FIG. 30 shows confocal photomicrographs of six-day 3D co-cultures of PAE(red) and leiomyosarcoma HTB-88 core biopsy xenograft (blue dashedellipses). Peripheral (top) or central (bottom) xenograft tissue wascultured with PAE. Co-cultures were incubated with (left) or without(right) Avastin®.

FIG. 31 shows a series of confocal photomicrographs of five-day 3Dco-cultures of HMEC-1 (yellow) and human leiomyosarcoma HTB-88 corebiopsy xenograft (blue dashed ellipses). Peripheral ringlet xenografttissue/HMEC-1 co-cultures were incubated with Avastin®, Thalidomide,Sunitinib or Fumagilin.

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS I. Abbreviations

2D: two-dimensional

3D: three-dimensional

ATCC: American Type Culture Collection

bFGF: basic fibroblast growth factor

BEC: brain endothelial cells

BME: Basement Membrane Extract

DMSO: dimethyl sulfoxide

EC50: The term half maximal effective concentration

EBM-2: endothelial basal medium-2

FBS: fetal bovine serum

FDA: Food and Drug Administration

GFP: green fluorescent protein.

HMEC-1: human microvascular endothelial cell

IPF: Idiopatic Pulmonary Fibrosis

LEC: lymphatic endothelial cells

PAE: porcine aortic endothelial cells

PBS: phosphate buffered saline

RFP: red fluorescent protein

VEGF: endothelial growth factor

YFP: yellow fluorescent protein

II. Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology canbe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. Although methodsand materials similar or equivalent to those described herein can beused in the practice or testing of this disclosure, suitable methods andmaterials are described below. The term “comprises” means “includes.”The abbreviation, “e.g.” is derived from the Latin exempli gratia, andis used herein to indicate a non-limiting example. Thus, theabbreviation “e.g.” is synonymous with the term “for example.”

To facilitate review of the various embodiments of the disclosure, thefollowing explanations of specific terms are provided:

Animal: A living multi-cellular vertebrate organism, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects, for example, humans, non-human primates,dogs, cats, horses, pigs, rats, mice, and cows.

Angiogenesis: A biological process leading to the generation of newblood vessels through sprouting and/or growth from pre-existing bloodvessels. The process can involve the migration and proliferation ofendothelial cells from preexisting vessels. Angiogenesis occurs duringpre-natal development, post-natal development, and in the adult. In theadult, angiogenesis occurs during the normal cycle of the femalereproductive system, wound healing, and during pathological processessuch as cancer (for a review see Battegay, J. Molec. Med. 73(7):333-346, 1995).

Angiogenic activity: The ability of an agent to promote or inhibitangiogenesis. Angiogenic activity can be measured in an angiogenesisassay, for example using the fluorescent cell lines and assays disclosedherein.

Angiogenic factor: A molecule that affects angiogenesis, for example bystimulating or inhibiting angiogenesis. Numerous experiments havesuggested that tissues secrete factors that promote angiogenesis underconditions of poor blood supply during normal and pathologicalangiogenesis processes. The formation of blood vessels is initiated andmaintained by a variety of factors secreted either by a cell (such as atumor cell) or by accessory cells. Many different growth factors andcytokines have been shown to exert chemotactic, mitogenic, modulatory orinhibitory activities on endothelial cells, smooth muscle cell andfibroblasts and can, therefore, be expected to participate in anangiogenic process. For example, factors modulating growth, chemotacticbehavior and/or functional activities of vascular endothelial cellsinclude aFGF, bFGF, angiogenin, angiotropin, epithelial growth factor,IL-8, and vascular endothelial growth factor (VEGF) among others.

Because many angiogenic factors are mitogenic and chemotactic forendothelial cells, their biological activities (such as angiogenicactivities) can be determined in vitro by measuring the inducedmigration of endothelial cells or the effect of these factors onendothelial cell proliferation using the cell lines assays and methodsdisclosed herein. For example, migration assays and other assays, suchas tubule formation assays and growth assays can also be used todetermine angiogenic activity, for example the angiogenic activity inthe presence of a test agent, such as a potential angiogenesisinhibitor.

Angiogenic potential: The ability of a factor, such as a compound orcell type, such as a tumor cell, to stimulate angiogenesis in anendothelial cell line.

Angiotropism: The movement of a tumor cell along a vascular highway.Such movement is a hallmark of metastasis of a tumor. In particularexamples, angiotropism is observable as the migration of tumor cells invitro along endothelial tubules.

Biological sample: A sample obtained from a plant or animal subjectabout which information is desired, for example, information about thesamples ability to promote cellular growth, tubule formation, and/orcellular migration. As used herein, biological samples include allclinical samples, including, but not limited to, cells, tissues, andbodily fluids, such as: blood; derivatives and fractions of blood, suchas serum, and lymphocytes (such as B cells, T cell, and subfractionsthereof); extracted galls; biopsied or surgically removed tissue,including tissues that are, for example, unfixed, frozen, fixed informalin and/or embedded in paraffin; tears; milk; skin scrapes; surfacewashings; urine; sputum; cerebrospinal fluid; prostate fluid; pus; bonemarrow aspirates; middle ear fluids, bronchoalveolar levage, trachealaspirates, sputum, nasopharyngeal aspirates, oropharyngeal aspirates, orsaliva. In particular embodiments, the biological sample is obtainedfrom an animal subject, such as in the form of middle ear fluids,bronchoalveolar levage, tracheal aspirates, sputum, nasopharyngealaspirates, oropharyngeal aspirates, or saliva. In particularembodiments, the biological sample is obtained from a subject, such asblood or serum. In other embodiments, the biological sample is a sampleof tissue removed from a tumor (e.g., a tumor biopsy). A patient sampleis a sample obtained from a subject, such as a mammalian subject, forexample a human subject under medical care.

Cellular activity: The activity of a particular cell line, such as theability of the cell to divide, migrate in response to stimulus, or toform three dimensional structures, such as tubules. The cellularactivity of a particular cell line can be assessed using in vitroassays, for example the assays disclosed herein.

Cancer: A malignant disease characterized by the abnormal growth anddifferentiation of cells. “Metastatic disease” refers to cancer cellsthat have left the original tumor site and migrate to other parts of thebody for example via the bloodstream or lymph system.

Examples of hematological tumors include leukemias, including acuteleukemias (such as acute lymphocytic leukemia, acute myelocyticleukemia, acute myelogenous leukemia and myeloblastic, promyelocytic,myelomonocytic, monocytic and erythroleukemia), chronic leukemias (suchas chronic myelocytic (granulocytic) leukemia, chronic myelogenousleukemia, and chronic lymphocytic leukemia), polycythemia vera,lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and highgrade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavychain disease, myelodysplastic syndrome, hairy cell leukemia, andmyelodysplasia.

Examples of solid tumors, such as sarcomas and carcinomas, includefibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy,pancreatic cancer, breast cancer (such as adenocarcinoma), lung cancers,gynecological cancers (such as, cancers of the uterus (e.g., endometrialcarcinoma), cervix (e.g., cervical carcinoma, pre-tumor cervicaldysplasia), ovaries (e.g., ovarian carcinoma, serous cystadenocarcinoma,mucinous cystadenocarcinoma, endometrioid tumors, celioblastoma, clearcell carcinoma, unclassified carcinoma, granulosa-thecal cell tumors,Sertoli-Leydig cell tumors, dysgerminoma, malignant teratoma), vulva(e.g., squamous cell carcinoma, intraepithelial carcinoma,adenocarcinoma, fibrosarcoma, melanoma), vagina (e.g., clear cellcarcinoma, squamous cell carcinoma, botryoid sarcoma), embryonalrhabdomyosarcoma, and fallopian tubules (e.g., carcinoma)), prostatecancer, hepatocellular carcinoma, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroidcarcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervicalcancer, testicular tumor, seminoma, bladder carcinoma, and CNS tumors(such as a glioma, astrocytoma, medulloblastoma, craniopharyogioma,ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,oligodendroglioma, menangioma, melanoma, neuroblastoma andretinoblastoma), and skin cancer (such as melanoma and non-melanoma).

Cell culture: The process by which either prokaryotic or eukaryoticcells are grown under controlled conditions. In practice the term “cellculture” has come to refer to the culturing of cells derived frommulticellular eukaryotes, especially animal cells, such as mammaliancells, for example the fluorescent cells disclosed herein. Mammaliancells are grown and maintained at an appropriate temperature and gasmixture (typically, 37° C., 5% CO₂) in a cell incubator. Cultureconditions vary widely for each cell type, and variation of conditionsfor a particular cell type can result in different phenotypes beingexpressed. Aside from temperature and gas mixture, the most commonlyvaried factor in culture systems is the growth medium. Recipes forgrowth media can vary in pH, glucose concentration, growth factors, andthe presence of other nutrient components. The growth factors used tosupplement media are often derived from animal blood, such as calfserum.

Some cells naturally live without attaching to a surface, such as cellsthat exist in the bloodstream. Others require a surface, such as mostcells derived from solid tissues. Cells grown unattached to a surfaceare referred to as suspension cultures. Other adherent cultures cellscan be grown on tissue culture plastic, which may be coated withextracellular matrix components (for example collagen or fibronectin) toincrease its adhesion properties and provide other signals needed forgrowth. Co-culture refers to the culture of more than one cell line(such as more than one of the disclosed cell lines), more than one celltype, or a cell line and a tissue sample, such a sample of a tumorbiopsy, in a single vessel. A 2-Dimensional (2D) co-culture is aco-culture wherein the different cell lines or cell types are notcultured within the same dimension or layer of the culture, and areseparated for example, by a gelled layer of gel matrix. A 3-Dimensional(3D) co-culture is a co-culture wherein the different cell lines or celltypes are cultured together within a three-dimensional gel matrix.

Chemical stimulus: A chemical signal that stimulates an activity of acell, or cell line, for example the cell lines disclosed herein.Examples of chemical stimuli include growth factors, such as bFGF andVEGF.

Chemotherapeutic agents: Any chemical agent with therapeutic usefulnessin the treatment of diseases characterized by abnormal cell growth. Suchdiseases include tumors, neoplasms, and cancer as well as diseasescharacterized by hyperplastic growth such as psoriasis. In oneembodiment, a chemotherapeutic agent is an angiogenesis inhibitor.Chemotherapeutic agents are described for example in Slapak and Kufe,Principles of Cancer Therapy, Chapter 86 in Harrison's Principles ofInternal Medicine, 14th edition; Perry et al., Chemotherapy, Ch. 17 inAbeloff, Clinical Oncology 2nd ed., 2000 Churchill Livingstone, Inc;Baltzer and Berkery. (eds): Oncology Pocket Guide to Chemotherapy, 2nded. St. Louis, Mosby-Year Book, 1995; Fischer Knobf, and Durivage (eds):The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book,1993. Combination chemotherapy is the administration of more than oneagent to treat cancer, for example an alkylating agent and anangiogenesis inhibitor.

Contacting: The placement in direct physical association, including bothin solid and in liquid form. Contacting can occur in vivo, for exampleby administering an agent to a subject, or in vitro for example withisolated cells or cell-cultures, for example cell-cultures of thedisclosed fluorescent cell lines. “Administrating” to a subject includestopical, parenteral, oral, intravenous, intra-muscular, sub-cutaneous,inhalational, nasal, or intra-articular administration, among others.

Control: A reference standard. A control can be a known value indicativeof basal cellular activity, such as basal migratory potential, doublingtime, tubule formation potential and the like, or a controlcell-culture, such as a culture including at least one of the disclosedfluorescent cell lines, not treated with an exogenous agent, such as atest agent, one or more cell lines (such as the fluorescent cell linesdisclosed herein), angiogenic factor, angiogenic inhibitor, or the like.A difference between a test sample and a control can be an increase orconversely a decrease. The difference can be a qualitative difference ora quantitative difference, for example a statistically significantdifference. In some examples, a difference is an increase or decrease,relative to a control, of at least about 10%, such as at least about20%, at least about 30%, at least about 40%, at least about 50%, atleast about 60%, at least about 70%, at least about 80%, at least about90%, at least about 100%, at least about 150%, at least about 200%, atleast about 250%, at least about 300%, at least about 350%, at leastabout 400%, at least about 500%, or greater then 500%.

Disperse: Distribute throughout a medium, such as a gel matrix of thedisclosed 3D co-cultures. In particular examples, cells that aredispersed in a medium are distributed evenly throughout the medium.However, dispersal of cells in a medium does not require absolute evendistribution of cells.

EC50: The term half maximal effective concentration (EC50) refers to theconcentration of a drug which induces a response halfway between thebaseline and maximum. EC50 is commonly used as a measure of drugpotency.

Encoding: Unless evident from its context, includes nucleic acidsequences, such as RNA and DNA sequences, that encode a polypeptide, aswell as RNA and DNA sequences that are transcribed into proteins, suchas fluorescent proteins, for example nucleic acid sequences that encodegreen fluorescent protein, red fluorescent protein, yellow fluorescentprotein, cyan fluorescent protein and the like.

Electromagnetic radiation: A series of electromagnetic waves that arepropagated by simultaneous periodic variations of electric and magneticfield intensity, and that includes radio waves, infrared, visible light,ultraviolet light, X-rays and gamma rays. In particular examples,electromagnetic radiation is emitted by a laser, which can possessproperties of monochromaticity, directionality, coherence, polarization,and intensity. Lasers are capable of emitting light at a particularwavelength (or across a relatively narrow range of wavelengths), forexample such that energy from the laser can excite one fluorophore witha specific excitation wavelength but not excite a second fluorophorewith a specific excitation wavelength difference and distinct from theexcitation wavelength on the first fluorophore.

Emission or emission signal: The light of a particular wavelengthgenerated from a source. In particular examples, an emission signal isemitted from a fluorophore, such as a fluorescent protein, after thefluorophore absorbs light at its excitation wavelength(s).

Excitation or excitation signal: The light of a particular wavelengthnecessary and/or sufficient to excite an electron transition to a higherenergy level. In particular examples, an excitation is the light of aparticular wavelength necessary and/or sufficient to excite afluorophore, such as a fluorescent protein, to a state such that thefluorophore will emit a different (such as a longer) wavelength of lightthen the wavelength of light from the excitation signal.

Exogenous agent: An exogenous agent is any agent external to a targetcell line(s) that is to be studied, and it includes small molecules,proteins, biological samples (such as patient samples) and other cellsor cell lines, such as fluorescent cell lines other than the target cellline, for example a different type of cell that can by identified asdifferent by a distinguishable fluorescent signal. In particularexamples, the exogenous agent is a test agent such as a small molecule,protein or nucleic acid, but which is not a cell or tissue sample.

Expression: With respect to a gene sequence, refers to transcription ofthe gene and, as appropriate, translation of the resulting mRNAtranscript to a protein. Thus, expression of a protein coding sequence,such as the expression of a fluorescent protein, results fromtranscription and translation of the coding sequence for that protein.Constitutive expression refers to the expression of a gene product, suchas a protein, for example a fluorescent protein, in a substantialcontinuous manner, such that the expression is not interrupted. Anexample of constitutive expression is continuous expression in theabsence of an exogenous stimulating agent, such as an agent used toactivate a promoter. Stable expression refers to expression that is notlost or reduced substantially over time, for example expression thatdoes not diminish through multiple passages of a cell line, for examplea cell line constitutively expressing a fluorescent protein.

Expression control sequences: Nucleic acid sequences that regulate theexpression of a heterologous nucleic acid sequence to which it isoperatively connected. Expression control sequences are operativelyconnected to a nucleic acid sequence when the expression controlsequences control and regulate the transcription and, as appropriate,translation of the nucleic acid sequence. Thus expression controlsequences can include appropriate promoters, enhancers, transcriptionterminators, a start codon (i.e., ATG) in front of a protein-encodinggene, splicing signal for introns, maintenance of the correct readingframe of that gene to permit proper translation of mRNA, and stopcodons. The term “control sequences” is intended to include, at aminimum, components whose presence can influence expression, and canalso include additional components whose presence is advantageous, forexample, leader sequences and fusion partner sequences. Expressioncontrol sequences can include a promoter.

A promoter is a minimal sequence sufficient to direct transcription.Also included are those promoter elements which are sufficient to renderpromoter-dependent gene expression controllable for cell-type specific,tissue-specific, or inducible by external signals or agents; suchelements may be located in the 5′ or 3′ regions of the gene. Bothconstitutive and inducible promoters are included (see e.g., Bitter etal., Methods in Enzymology 153:516-544, 1987). For example, when cloningin bacterial systems, inducible promoters such as pL of bacteriophagelambda, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may beused. In one embodiment, when cloning in mammalian cell systems,promoters derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g., theretrovirus long terminal repeat; the adenovirus late promoter; thevaccinia virus 7.5K promoter; the SV40 viral promoter; the CMV promoterand the like) can be used. Promoters produced by recombinant DNA orsynthetic techniques may also be used to provide for transcription ofthe nucleic acid sequences, for example when incorporated into a vector,such as a mammalian expression vector.

Fluorescent property: A characteristic of a fluorescent molecule, suchas a fluorescent protein, for example green fluorescent protein, redfluorescent protein, yellow fluorescent protein, cyan fluorescentprotein and the like. Examples of fluorescent properties include themolar extinction coefficient at an appropriate excitation wavelength,the fluorescence quantum efficiency, the shape of the excitationspectrum or emission spectrum (the “fluorescence spectrum,” theexcitation wavelength maximum and emission wavelength maximum, the ratioof excitation amplitudes at two different wavelengths, the ratio ofemission amplitudes at two different wavelengths, the excited statelifetime, or the fluorescence anisotropy. Quantifying fluorescencerefers to the determination of the amount of fluorescence generated by afluorophore, for example a fluorescent protein, which can be thequantity of photons emitted by a fluorophore. In some examples,fluorescence is quantified by measuring the intensity of a fluorescencesignal at a particular wavelength, for example the wavelength of theemission maxima of a particular fluorophore, such as a fluorescentprotein. Fluorescence intensity can also be quantified at a wavelengththat is not the emission maxima of a particular fluorophore, for exampleto avoid emission spectra that overlap and thereby interfere with theemission maxima of a particular fluorophore, such as a particularfluorescent protein. In some examples, a fluorescence signal is emittedby a population of fluorescent proteins, for example fluorescentproteins present in a population of cells containing such fluorescentproteins. Such a signal can be quantified, for example to determine thenumber, or relative number of cells that emit such a fluorescent signal.Detecting a pattern of fluorescence refers to the correlation of afluorescent signal to a specific location to determine the locationwhere a fluorescence signal, such as a fluorescent signal of aparticular wavelength, originates. In some examples, a pattern offluorescence determines the location and or shape of the cells that emita fluorescence signal, such as cells containing a fluorescent protein,for example to determine the number of the total area of the tubules,the total number of tubules, number of nodes, number of branch points,the number of tubes per node, or node area formed by such cells usingthe methods disclosed herein.

Fluorescent protein: A protein capable of emission of a detectablefluorescent signal. Fluorescent proteins can be characterized by thewavelength of their emission spectrum. For example green fluorescentprotein (GFP) has a fluorescent emission spectrum in the green part ofthe visible spectrum. In addition to green-fluorescent proteins,fluorescent proteins are known which fluoresce in other regions of thevisible spectrum, for example blue-fluorescent proteins,cyan-fluorescent proteins, yellow-fluorescent proteins,orange-fluorescent proteins, red-fluorescent proteins, and far-redfluorescent proteins. Examples of fluorescent proteins can be found inthe following patent documents: U.S. Pat. Nos. 5,804,387; 6,090,919;6,096,865; 6,054,321; 5,625,048; 5,874,304; 5,777,079; 5,968,750;6,020,192; 6,146,826; 6,969,597; 7,150,979; 7,157,565; and 7,166,444;and published international patent applications WO 07/085,923; WO07/052,102, WO 04/058973, WO 04/044203, WO 03/062270; and WO 99/64592.Additional examples of fluorescent proteins are available from Clontech,Laboratories, Inc. (Mountain View, Calif.) under the trade name LivingColors®. Nucleic acids encoding such fluorescent proteins can beincorporated into mammalian expression vectors for use in producing thedisclosed fluorescent cell lines.

Gel Matrix: A semi-solid cell culture media that is derived fromextracellular matrix proteins or any suitable equivalent synthetic gelproduct. Gel matrices are fluid at 4° C. and gel at 37° C. In particularexamples a gel matrix is a commercially available medium such as BDMatrigel™ Matrix (BD Bioscience), Cultrex® BME (Trevigen), or Geltrex®(Invitrogen®). Other basement membrane extracts that can function as asupport matrix scaffolding include human placenta-derived BME (VivoBiosciences, Inc) and synthetic BME (available from GlycosanBiosystems).

Gene: A nucleic acid sequence that encodes a polypeptide under thecontrol of a regulatory sequence, such as a promoter or operator. A geneincludes an open reading frame encoding a polypeptide of the presentdisclosure, as well as exon and (optionally) intron sequences. An intronis a DNA sequence present in a given gene that is not translated intoprotein and is generally found between exons. The coding sequence of thegene is the portion transcribed and translated into a polypeptide (invivo, in vitro or in situ) when placed under the control of anappropriate regulatory sequence. The boundaries of the coding sequencecan be determined by a start codon at the 5′ (amino) terminus and a stopcodon at the 3′ (carboxyl) terminus. If the coding sequence is intendedto be expressed in a eukaryotic cell, a polyadenylation signal andtranscription termination sequence can be included 3′ to the codingsequence.

Transcriptional and translational control sequences include, but are notlimited to, DNA regulatory sequences such as promoters, enhancers, andterminators that provide for the expression of the coding sequence, suchas expression in a host cell. A polyadenylation signal is an exemplaryeukaryotic control sequence. A promoter is a regulatory region capableof binding RNA polymerase and initiating transcription of a downstream(3′ direction) coding sequence.

Growth rate: The expansion of the number of cells of a specified cellline through cell division as a function of time. In one example thegrowth rate is the rate at which a cell line grown in culture doubles.

Preferred mammalian codon(s): The subset of codons from among the set ofall possible codons encoding an amino acid that are most frequently usedin proteins expressed in mammalian cells. Table 1 summarizes preferredmammalian codons for each amino acid:

TABLE 1 Amino Acid Preferred codons Gly GGC, GGG Glu GAG Asp GAC ValGUG, GUC Ala GCC, GCU Ser AGC, UCC Lys AAG Asn AAC Met AUG Ile AUC ThrACC Trp UGG Cys UGC Tyr UAU, UAC Leu CUG Phe UUC Arg CGC, AGG, AGA GlnCAG His CAC Pro CCCIn some embodiments, the nucleotide sequence encoding the amino acidsequence of a fluorescent protein has been codon optimized forexpression in a mammalian cell. By codon optimized it is meant that atleast some of the codons that encode the fluorescent protein have beenexchanged for codons that are preferentially used by mammalian cells,for example the codons listed in Table 1. Typically the exchange ofcodons does not alter the amino acid sequence of the resultingfluorescent protein relative to the fluorescent protein encoded by thenucleic acid sequence with unexchanged codons.

High throughput technique: Through this process one can rapidly identifyactive compounds, antibodies or genes which affect a particularbiomolecular pathway, for example pathways in angiogenesis. In certainexamples, combining modern robotics, data processing and controlsoftware, liquid handling devices, and sensitive detectors, highthroughput techniques allows the rapid screening of potentialpharmaceutical agents in a short period of time.

Histology: The study of the microscopic anatomy and classification oftissue, including the histology of mammalian cells, such as cells andcell lines from mammalian tissues. Histological typing refers to thecategorizing of tissue into histological types, for example bymicroanatomical origin (such as connective tissue, nerves, muscles, andcirculatory cells, among others) or cell-types (such as epithelialcells, stromal cells among others). Cells can be classified as being ofdifferent histological types by virtue of the staining and/or reactionwith antibodies, or by characteristic microanatomical features. Cells ofdifferent histological types interact differently with different stainsand/or antibodies. Methods for histological typing are well known in theart. Histology can be use to determine if cells are of different types.Thus, in some examples different cell lines are histologically differentcell lines.

Immortalized cell or cell line: A cell or cell line that has acquiredthe ability to proliferate indefinitely either through random mutationor deliberate modification, such as artificial expression of thetelomerase gene. There are numerous well established immortalized celllines representative of particular cell types.

Inhibitor (for example, of angiogenesis): A substance capable ofinhibiting to some measurable extent, for example angiogenesis. Indisclosed examples inhibition is measured in the assays disclosedherein.

Isolated: An “isolated” biological component (such as a cell (or cellline), nucleic acid, peptide or protein) has been substantiallyseparated, produced apart from, or purified away from other biologicalcomponents in the cell of the organism in which the component naturallyoccurs, for instance, other chromosomal and extrachromosomal DNA andRNA, and proteins. Nucleic acids, peptides and proteins that have been“isolated” thus include nucleic acids and proteins purified by standardpurification methods. The term also embraces nucleic acids, peptides andproteins prepared by recombinant expression in a cell as well aschemically synthesized nucleic acids. The term “isolated” or “purified”does not require absolute purity; rather, it is intended as a relativeterm. Thus, for example, an isolated peptide preparation is one in whichthe peptide or protein is more enriched than the peptide or protein isin its natural environment within a cell. Preferably, a preparation ispurified such that the protein or peptide represents at least 50% of thetotal peptide or protein content of the preparation. In addition, theterm “isolated” can also be applied to a cell or a cell line, forexample an isolated cell or cell line is one that is removed from itsoriginal host. Isolated cells or cell lines can be placed back in ahost, even the host from which they were originally isolated.

Metastatic potential: The ability of cancer cells to leave the originaltumor site and migrate to other parts of the body, for example via thebloodstream or lymphatic system. In particular examples, the metastaticpotential of a tumor cell is indicated by movement of a tumor cell alongpre-vascular endothelial tubules in vitro (angiotropism).

Migration potential: The ability of cells, such as the cell linedisclosed herein, to translocate in response to a chemical stimulus,such as a growth factor. Migration potential can be determined with theassays disclosed herein.

Mimetic: The ability for a composition or an environment to resembleanother composition or environment. In particular examples, an in vitrocell culture provides a mimetic to an in vivo context when the cells ofthe culture behave in a manner that correlates to their in vivobehavior.

Mixed cell population: A population of cells, such as cells in culture,that contains two or more different types of cells, such ashistologically different cell lines. Examples of different types ofcells include cells of different embryonic origin (such as cellsoriginating from the ectoderm, endoderm, or mesoderm), cells fromdifferent cellular locations (such as cells from epithelium,endothelium, or stroma), cells from different tissues or organs (such ascells from pulmonary myocardial, neural, vascular, skin, bone, orskeletal or smooth muscle tissue).

Neoplasm or tumor: Any new and abnormal growth; particularly a newgrowth of tissue in which the growth is uncontrolled and progressive. Aneoplasm, or tumor, serves no useful function and grows at the expenseof the healthy organism.

In general, tumors appear to be caused by abnormal regulation of cellgrowth. Typically, the growth of cells in the body is strictlycontrolled; new cells are created to replace older ones or to performnew functions. If the balance of cell growth and death is disturbed, atumor may form. Abnormalities of the immune system, which usuallydetects and blocks aberrant growth, also can lead to tumors. Othercauses include radiation, genetic abnormalities, certain viruses,sunlight, tobacco, benzene, certain poisonous mushrooms, and aflatoxins.

Tumors are classified as either benign (slow-growing and usuallyharmless depending on the location), malignant (fast-growing and likelyto spread and damage other organs or systems) or intermediate (a mixtureof benign and malignant cells). Some tumors are more common in men orwomen, some are more common amongst children or elderly people, and somevary with diet, environment and genetic risk factors.

Symptoms of neoplasms depend on the type and location of the tumor. Forexample, lung tumors can cause coughing, shortness of breath, or chestpain, while tumors of the colon can cause weight loss, diarrhea,constipation and blood in the stool. Some tumors produce no symptoms,but symptoms that often accompany tumors include fevers, chills, nightsweats, weight loss, loss of appetite, fatigue, and malaise.

Blood vessels supply tumors with nutrients and oxygen. Tumor growth isdependent on the generation of new blood vessels that can maintain theneeds of the growing tumor, and many tumors secrete substances(angiogenic factors) that are able to induce proliferation of new bloodvessels (angiogenesis). Anti-tumor therapies include the use ofangiogenesis inhibitors, which reduce the formation of blood vessels inthe tumor, effectively starving the tumor and/or cause the tumor todrown in its own waste.

Neovascularization: The growth of new blood vessels. Neovascularizationcan be the proliferation of blood vessels in tissue not normallycontaining them, or the proliferation of blood vessels in an ischemic orotherwise damaged tissue. Neovascularization can be pathological when itis unwanted or mediates a pathological process, for example when itoccurs in the retina or cornea.

Neutral: A molecule is neutral when its overall charge is neitherpositive nor negative. One example of a neutral polysaccharide isagarose.

Nucleic acid: A deoxyribonucleotide or ribonucleotide polymer in eithersingle (ss) or double stranded (ds) form, and can include analogues ofnatural nucleotides that hybridize to nucleic acids in a manner similarto naturally occurring nucleotides. In some examples, a nucleic acid isa nucleotide analog.

Unless otherwise specified, any reference to a nucleic acid moleculeincludes the reverse complement of nucleic acid. Except wheresingle-strandedness is required by the text herein (for example, a ssRNAmolecule), any nucleic acid written to depict only a single strandencompasses both strands of a corresponding double-stranded nucleicacid. For example, depiction of a plus-strand of a dsDNA alsoencompasses the complementary minus-strand of that dsDNA. Additionally,reference to the nucleic acid molecule that encodes a specific protein,or a fragment thereof, encompasses both the sense strand and its reversecomplement.

Operably connected or operably linked: A first nucleic acid sequence isoperably connected to a second nucleic acid sequence when the firstnucleic acid sequence is placed in a functional relationship with thesecond nucleic acid sequence. For instance, a promoter is operablyconnected to a coding sequence, such as the coding sequence of afluorescent protein, for example GFP, if the promoter affects thetranscription or expression of the coding sequence. Generally, operablyconnected DNA sequences are contiguous.

Passaging cells: Passaging or splitting cells involves transferring asmall number of cells into a new vessel. Cells can be cultured for alonger time if they are split regularly, as it avoids the senescenceassociated with prolonged high cell density. Suspension cultures areeasily passaged with a small amount of culture containing a few cellsdiluted in a larger volume of fresh media. For adherent cultures, cellsfirst need to be detached; which is typically done with a mixture oftrypsin-EDTA. A small number of detached cells can then be used to seeda new culture.

Pharmaceutical agent or drug: A chemical compound or composition capableof inducing a desired therapeutic or prophylactic effect when properlyadministered to a subject (such as the inhibition of angiogenesis),alone or in combination with another therapeutic agent(s) orpharmaceutically acceptable carriers. Pharmaceutical agents include, butare not limited to, angiogenic factors, for example bFGF, and VEGF, andanti-angiogenic factors, such as inhibitors of bFGF, or VEGF. Forexample, suitable anti-angiogenic factors include, but are not limitedto, SU5416, which is a specific VEGF-R antagonist, SU6668 which blocksthe receptors for VEGF, bFGF, and PDGF and Avastin®. See, for example,Liu et al., Seminars in Oncology 29 (Suppl 11): 96-103, 2002; Shepherdet al., Lung Cancer 34:S81-S89, 2001.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers of use are conventional. Remington's Pharmaceutical Sciences,by E.W. Martin, Mack Publishing Co., Easton, Pa., 15th Edition, 1975,describes compositions and formulations suitable for pharmaceuticaldelivery of the compositions disclosed herein.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (such as powder, pill, tablet, orcapsule forms), conventional non-toxic solid carriers can include, forexample, pharmaceutical grades of mannitol, lactose, starch, ormagnesium stearate. In addition to biologically neutral carriers,pharmaceutical compositions to be administered can contain minor amountsof non-toxic auxiliary substances, such as wetting or emulsifyingagents, preservatives, and pH buffering agents and the like, for examplesodium acetate or sorbitan monolaurate.

Primary cells: Cells that are cultured directly from a subject. With theexception of some derived from tumors, most primary cell cultures havelimited lifespan. After a certain number of population doublings cellsundergo the process of senescence and stop dividing, while generallyretaining viability.

Protein coding sequence or a sequence that encodes a peptide: A nucleicacid sequence that is transcribed (in the case of DNA) and is translated(in the case of mRNA) into a peptide in vitro or in vivo when placedunder the control of appropriate regulatory sequences. The boundaries ofthe coding sequence are determined by a start codon at the 5′ (amino)terminus and a translation stop codon at the 3′ (carboxy) terminus. Acoding sequence can include, but is not limited to, cDNA fromprokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryoticor eukaryotic DNA, and even synthetic DNA sequences. A transcriptiontermination sequence is usually located 3′ to the coding sequence.

Signal: A detectable change or impulse in a physical property thatprovides information. In the context of the disclosed methods, examplesinclude electromagnetic signals, such as light, for example light of aparticular quantity or wavelength, for example a wavelength of lightemitted from a fluorescent protein.

Recombinant: A recombinant nucleic acid or protein is one that has asequence that is not naturally occurring (for example a vector, such asa vector encoding a fluorescent protein, such as GFP and the like) orhas a sequence that is made by an artificial combination of twootherwise separated segments of sequence. This artificial combinationcan be accomplished, for example, by chemical synthesis or by theartificial manipulation of isolated segments of nucleic acids orproteins, for example, by genetic engineering techniques.

Test agent: Any agent that that is tested for its effects, for exampleits effects on a cell. In some embodiments, a test agent is a chemicalcompound, such as a chemotherapeutic agent or even an agent with unknownbiological properties.

Therapeutically effective amount: A dose sufficient to have atherapeutic effect, for example to inhibit to some degree advancement,or to cause regression of the disease, or which is capable of relievingsymptoms caused by the disease. For example, a therapeutically effectiveamount of an angiogenesis inhibitor can vary from about 0.1 nM perkilogram (kg) body weight to about 1 μM per kg body weight, such asabout 1 nM to about 500 nM per kg body weight, or about 5 nM to about 50nM per kg body weight. The exact dose is readily determined by one ofskill in the art based on the potency of the specific compound, the age,weight, sex and physiological condition of the subject.

Treating: Inhibiting the full development of a disease or condition, forexample, in a subject who is at risk for a disease such as cancer.“Treatment” refers to a therapeutic intervention that ameliorates a signor symptom of a disease or pathological condition after it has begun todevelop. The term “ameliorating,” with reference to a disease orpathological condition, refers to any observable beneficial effect ofthe treatment. The beneficial effect can be evidenced, for example, by adelayed onset of clinical symptoms of the disease in a susceptiblesubject, a reduction in severity of some or all clinical symptoms of thedisease, a slower progression of the disease, an improvement in theoverall health or well-being of the subject, or by other parameters wellknown in the art that are specific to the particular disease. A“prophylactic” treatment is a treatment administered to a subject whodoes not exhibit signs of a disease or exhibits only early signs for thepurpose of decreasing the risk of developing pathology. A personalizedtreatment is a treatment that is tailored to a particular subject basedon the characteristics of the subject and optionally also the particulardisease.

Transduced Transformed, Transfected: A virus or vector “transduces” or“transfects” a cell when it transfers nucleic acid into the cell. A cellis “transformed” by a nucleic acid transduced into the cell when the DNAbecomes stably replicated by the cell, either by incorporation of thenucleic acid into the cellular genome, or by episomal replication. Asused herein, the term transformation encompasses all techniques by whicha nucleic acid molecule might be introduced into such a cell, includingtransfection with viral vectors, transformation with plasmid vectors,and introduction of naked DNA by electroporation, lipofection, andparticle gun acceleration.

Tumor spheroid colony: An in vitro clonal expansion of a single parentaltumor cell and which has the three dimensional characteristics of an invivo tumor. Tumor spheroids include not only morphogenic capacities andhistotypic reorganization of an in vivo tumor, but also maintain itsfunctional activities and gene expression patterns (Hauptmann et al.,Int. J. Cancer. 61:819-825, 1995). Additionally, tumor spheroids providea simple geometry for modeling the effects of anticancer treatments(Buffa et al., Int J Radiat Oncol Biol Phys. 49:1109-1118, 2001).

Tumor biopsy: A section of tumor tissue removed from a whole tumor, forexample a tumor from a subject. Tumor biopsies contain a mixedpopulation of cells including tumor and stromal cells. In particularexamples, a tumor biopsy is a tumor tissue plug, which is a section of apunch biopsy (periphery, mid-section, and central core) from a tumor,such as a tumor from a subject or a xenograft tumor.

Tubule formation potential: The ability of a cell line to form atube-like structure in vitro, for example a structure similar to a bloodvessel, such as a capillary. Tubule formation potential can bedetermined by determining the pattern displayed by cells which have beeninduced to form tubules, for example by determining the pattern offluorescence from cells expressing fluorescent proteins, such as thecell lines disclosed herein. In particular examples, tubule formationpotential is a characteristic of endothelial cells. In other examples itis a characteristic of other cell types including certain tumor cellslines (e.g. MDA-MB-435) and pericytes.

Vector: A nucleic acid molecule that can be introduced into a cell,thereby producing a transformed cell. A vector can include nucleic acidsequences that permit it to replicate in a cell, such as an origin ofreplication, for example a SV40 origin for replication in mammaliancells and a pUC origin of replication for propagation in E. coli, andcan also include one or more selectable marker genes, such as antibioticresistance genes, such as the kanamycin resistance gene and the neomycinresistance gene. Other genetic elements and protein coding sequences canalso be included in the vector, such as sequences encoding a fluorescentprotein, for example GFP or the like, promoters for the expression ofproteins, such as the SV40 early promoter and the immediate earlypromoter of cytomegalovirus (PCMV IE), Kozak translation initiationsequences, and polyadenylation signals, such as the SV40 polyadenylationsignal.

III. Description of Several Embodiments

Understanding biological processes that underlie cellular organization,such as in organ development and the pathogenesis of diseases such ascancer, would be facilitated by methods for studying these complexinteractions in vitro. In vitro assays are needed to investigate therelationship between multiple cellular components involved in thebiological processes (such as angiogenesis), investigate newcombinatorial approaches to boost the efficiency of existingtherapeutics, and to facilitate the discovery of new potential singleand/or combination drugs. Disclosed herein is an in vitro assay thatmeets these needs.

A basic component of this in vitro assay is the immortalized fluorescentcell lines disclosed herein. The disclosed fluorescent cell linesrepresent an array of different cell types, such as cell types thatcontribute to biological processes, such as angiogenesis, in vivo. Thedisclosed fluorescent cell lines are derived from different anatomicalorigins which are known to be relevant during the angiogenesis process.In particular examples, the cells are mammalian cell lines, such ashuman cell lines, and may be immortalized cell lines. The disclosed celllines have been stably transfected with mammalian expression plasmidsthat constitutively express different fluorescent proteins, for examplegreen fluorescent protein and related florescent proteins, such asyellow fluorescent protein, red fluorescent protein, cyan fluorescentprotein and the like.

The fluorescence signals produced by the cell lines expressing greenfluorescent protein and related fluorescent proteins can be used forreal time direct estimation of cell numbers, because the fluorescentsignal from a population of cells stably and constitutively expressing afluorescent protein is proportional to the number of such cells. Inaddition, because these fluorescent cell lines emit a detectable signalthat can be localized in space, the individual cells can be localized inspace, for example to determine a pattern of fluorescence attributableto the cells. These features can be used in a number of different assaysincluding growth assays, migration assays, tubule formation assays, cellviability assays, and the like. The use of fluorescent cells in suchassays, for example the assays disclosed herein, not only eliminates theneed for expensive commercially available kits (for example kits neededto generate and end point readable signal, for example a chemical agentthat renders the cell, or cell morphology detectable) but alsosimplifies procedures by considerably shortening the protocol time,because no additional agents need to be added to generate a signal.Additionally, because no detection reagents have to be added to thecells in culture the fluorescent cell lines and assays disclosed hereinavoided putatively harmful interactions between those chemicals and thecellular components under study.

Deregulation of angiogenesis plays a major role in a number of humandiseases. A dramatic increase in the research effort in the field ofangiogenesis has resulted in a substantial understanding of theangiogenic process and subsequently the development of new therapeuticsto modulate angiogenesis. Although angiogenesis inhibitors are among themost promising drug candidates for cancer, the existing “single drug,non-personalized” approach has proven to be problematic regardingextending patient survival time and the development of drug resistanttumor clones.

The inability to develop more successful therapies is hampered byinsufficient knowledge about the interactions between the multiplecellular components involved in the angiogenesis process and theinability to evaluate angiogenic potential in individual patients.Hence, one of the major problems confronting clinicians today is theability to assess angiogenic/antiangiogenic therapy effectiveness in amixed cell environment, such as the mixed cell environment responsiblefor angiogenesis. The disclosed assays are particularly suited to theinvestigation of angiogenesis especially in a mixed cell environment,such as a mixed cell environment approximating in vivo conditions (forexample a mixed cell population shown in FIG. 14).

The disclosed assays can be used to monitor the effects of a drug, suchas an existing angiogenesis inhibitor or other chemotherapeutic agent,on a patient sample, for example, by determining the effect of a patientsample, patient serum, plasma, tumor cells, on the fluorescent celllines disclosed herein. Using the disclosed assays, a patient samplefrom a patient with cancer could have elevated level of proangiogenicfactors, such as growth factors. Using the disclosed assays, it ispossible to monitor the patient, via monitoring a sample obtained from apatient, to determine if a particular treatment is having a desiredeffect, for example reducing the level of proangiogenic factors presentin the sample. Thus, a particular therapy can be developed for anindividual patient, for example a personalized combinatorial drugtherapy which would be effective for that individual.

Also disclosed herein are methods for monitoring angiogenic ormetastatic potential of tumor cells, the methods comprising preparing athree-dimensional co-culture that is comprised of three layers. Thefirst layer comprises a neutral polysaccharide polymer gel in contactwith the bottom of the culture dish. The second layer is on top of thefirst layer and comprises a solidified gel matrix, endothelial cellsthat are dispersed in the solidified gel matrix; and tumor cellscomprising either a tumor spheroid colony or a sample of a tumor biopsy,and which are also suspended in the solidified gel matrix, and a thirdlayer comprising culture medium. Angiogenic or metastatic potential oftumor cells is monitored by incubating the three-dimensional co-culture;and detecting at least one of endothelial cell proliferation,endothelial cell tubule formation or tumor cell angiotropism of thecells in the second layer. In particular examples, the neutralpolysaccharide polymer gel comprises agarose. In other examples, theendothelial cells stably and constitutively express a fluorescentprotein. In still other examples, the tumor cells stably andconstitutively express a fluorescent protein with a different emissionspectrum from the fluorescent protein expressed by the endothelialcells. In yet further examples, the second layer further comprises atleast one additional mammalian cell type dispersed in the solidified gelmatrix, such as a cell type selected from the group consisting ofmacrophage, mast cell, fibroblast, adipocyte, and pericyte. In stillfurther examples, the additional mammalian cell type stably andconstitutively expresses a fluorescent protein with a different emissionspectrum from the fluorescent protein expressed by the endothelialcells.

In particular examples of the disclosed methods for monitoringangiogenic or metastatic potential of tumor cells, the first, second, orthird layer further comprises at least one test agent, which in someexamples is a known or potential inhibitor or promoter of angiogenesisor metastasis.

In other examples of the disclosed methods, the tumor cells are derivedfrom a subject and the first, second, or third layer further comprisesat least one test agent that has been administered to the subject aspart of a cancer treatment.

Further disclosed herein are methods of testing the efficacy of ananti-angiogenic or anti-metastatic cancer treatment for a subject,comprising monitoring angiogenic or metastatic potential of tumor cellsby the above described methods utilizing a three dimensional co-culture,wherein the tumor cells are derived from the subject and the first,second, or third layer comprises at least one test agent that is acandidate anti-cancer treatment.

Additionally disclosed herein are methods of selecting a personalizedanti-angiogenic or anti-metastatic treatment for cancer in a subjectcomprising preparing multiple three-dimensional co-cultures, eachco-culture comprising a first layer comprising a neutral polysaccharidepolymer gel in contact with the bottom of a culture dish; a second layeron top of the first layer, comprising: a solidified gel matrix;endothelial cells dispersed in the solidified gel matrix; and tumorcells comprising either a tumor spheroid colony or a sample of a tumorbiopsy, suspended in the solidified gel matrix; and a third layer on topof the second layer comprising culture medium, wherein all but one ofthe co-cultures further comprises at least one test agent comprising ananti-angiogenic or anti-metastatic compound in the first, second, orthird layers. A personalized anti-angiogenic or anti-metastatictreatment for cancer in a subject is selected by incubating thethree-dimensional co-cultures; detecting at least one of endothelialcell proliferation, endothelial cell tubule formation or tumor cellangiotropism of the cells in the second layer; and selecting the atleast one test agent having the greatest effect on at least one ofendothelial cell proliferation, endothelial cell tubule formation ortumor cell angiotropism in comparison to endothelial cell proliferation,endothelial cell tubule formation or tumor cell angiotropism in thecells of the co-culture without the test agent. In particular examples,the neutral polysaccharide polymer gel comprises agarose. In someexamples, the endothelial cells stably and constitutively express afluorescent protein. In other examples, the tumor cells stably andconstitutively express a fluorescent protein with a different emissionspectrum from the fluorescent protein expressed by the endothelialcells. In particular examples, the second layer further comprises atleast one additional mammalian cell type dispersed in the solidified gelmatrix, which in some examples, stably and constitutively expresses afluorescent protein with a different emission spectrum from thefluorescent protein expressed by the endothelial cell, and in furtherexamples is a cell type selected from the group consisting ofmacrophage, mast cell, fibroblast, adipocyte, and pericyte.

A. Assays

Aspects of this disclosure relate to an assay method, such as amultiplex assay method, for evaluating cellular activity, for examplethe cellular activity of the disclosed fluorescent cell lines. Thedisclosed method involves providing an in vitro culture of one or morecell lines, such as an in vitro mixture of one or more of thefluorescent cell lines disclosed herein, for example cell lines ofdifferent histological types, for example different types of mammaliancells, such as mammalian somatic cells. Examples include porcine aorticendothelial cell line PAE, human lymphatic endothelial cell line LEC-1,human microvascular endothelial cell line HMEC-1, or rhesus macaquechoroidal endothelial cell line RF/6A (ATTC CRL-1780) (which has markercharacteristics of a pericyte line (SMA, TIMP-3, NG2, PDGFR-β, etc);epithelial cell lines, such as human adenocarcinoma cell line A549;adenocarcinoma cell lines, such as human breast adenocarcinoma cell lineMCF7; or mast cell cell lines, such as human mast cell line HMC-1, amongothers. These particular cell lines are examples of different cell linesbelieved to play a role in angiogenesis.

In some embodiments, an in vitro cell line mixture is provided whichcontains a first isolated mammalian cell line stably and constitutivelyexpressing a first fluorescent protein and one or more additionalisolated mammalian cell lines stably and constitutively expressingfluorescent proteins having an emission spectrum different from theemission spectrum of the first fluorescent protein and having differenthistological types. The in vitro cell line mixture is cultured and thecellular activity of the first isolated mammalian cell line or one ormore addition isolated mammalian cell lines present in the culture isassessed by quantifying fluorescence or detecting a pattern offluorescence from the first fluorescent protein or the fluorescentprotein expressed by the one or more additional mammalian cell lines. Byproviding a mixture of isolated mammalian cell lines of differenthistological types each expressing a different fluorescent protein withdifferent emission spectra, it is possible to assess the cellularactivity of the multiple cell lines present in the mixture, for examplesimultaneously or serially. Thus, in some embodiments, the cellularactivity of the first isolated mammalian cell line present in themixture is assessed and the cellular activity of the additional isolatedmammalian cell lines present in the cell line mixture is assessed byquantifying fluorescence or detecting a pattern of fluorescence from thefirst fluorescent protein and the fluorescent proteins present in theadditional isolated cell lines present in the culture.

In some embodiments, an in vitro cell line mixture is provided whichcontains a first isolated mammalian cell line stably and constitutivelyexpressing a first fluorescent protein and a second isolated mammaliancell line stably and constitutively expressing a second fluorescentprotein, in which the first and second fluorescent proteins havedifferent emission spectra and the first isolated mammalian cell lineand the second isolated mammalian cell line are different cell lines,for example cell lines of different histological types. In someembodiments, the cellular activity of the first isolated mammalian cellline present in the mixture is assessed by quantifying fluorescence (forexample by quantifying the fluorescence intensity at a particularwavelength, such as the emission maxima) or detecting a pattern offluorescence from the first fluorescent protein (such as the pattern offluorescence of the fluorescent proteins present in the fluorescent cellline, for example the location of the fluorescence in two or threedimensional space). In some embodiments, the cellular activity of thefirst isolated mammalian cell line present in the mixture is assessedand the cellular activity of the second isolated cell line present inthe cell line mixture is assessed by quantifying fluorescence ordetecting a pattern of fluorescence from the first fluorescent proteinand the second fluorescent protein.

In some embodiments, an in vitro cell line mixture is provided whichcontains at least three, such as three, four, five, six or more,different isolated mammalian cell lines, such as different cell lineseach having a different histological type, wherein each isolatedmammalian cell line stably and constitutively expresses a differentfluorescent protein having an emission spectrum distinguishable from theother fluorescent proteins. In such a mixture each cell line is uniquelyassociated with a particular fluorescent protein having a differentemission spectrum from the other fluorescent proteins so that theindividual cell lines present in the mixture can be distinguished, suchthat the fluorescence from the individual cell line can be quantifiedand/or the pattern of fluorescence detected. Thus, the quantifiedfluorescence or pattern of fluorescence attributable to a specificfluorescent cell line can be determined.

In some embodiments, assessing cellular activity includes determiningthe growth rate, migration potential, and/or tubule formation potentialof the first isolated mammalian cell line and/or additional isolatedcell lines present in the in vitro mixture using the quantifiedfluorescence or the pattern of fluorescence from the fluorescentproteins in the mixture, such as first fluorescent protein and/or theadditional fluorescent proteins, such as a second, third forth, fifth,six, etc. fluorescent proteins present in the in vitro mixture. In someexamples, the number of dead cells present in the mixture is determinedby determining the quantified fluorescence present in the media of thecell-mixture. Exemplary methods for determining the growth rate, celldeath, migration potential, or tubule formation potential of theisolated cell lines disclosed herein are given below.

i. Growth Assay

Using the disclosed fluorescent cell lines, a real time growth assay hasbeen developed and applied to mono- or multiple-cell cultures(co-culture). As disclosed herein the fluorescence signal emitted by aculture of the disclosed fluorescent cell lines is proportional to thenumber of fluorescent cells present in the culture (see for example FIG.2). In other words, the fluorescence signal, for example measured as theintensity of the emission maxima, from a population of fluorescent cellsof one type in a culture will double as the number of fluorescent cellsof that type in the culture doubles. Conversely, the fluorescencesignal, for example measured as the intensity of the emission maxima,from a population of cells of one type in a culture will be reduced tohalf if the number of cells of that type in the culture is divided inhalf. These properties can be used to measure the effect of an exogenousagent, such as one or more additional cell lines, or a test agent, onthe fluorescent cells in culture. It should be noted that at some pointthe total fluorescence of a culture may reach signal saturation, suchthat the signal reaches a plateau as a function of cell number.

In some embodiments, the effect of an additional cell line (for examplea different cell line) on a first fluorescent cell line is determined(this can be extended to multiple cell lines and even one or morefluorescent cell lines, or combinations thereof, for example in amultiplex assay). For example, as shown in FIG. 4, the effect of theyellow fluorescent PAE cell line on the red fluorescent MCF7 cell linehas been measured.

In some embodiments, the disclosed growth assay is used to assess if thepresence of one or more additional cell lines, such as one or more ofthe fluorescent cell lines disclosed herein, affects the growth rate ofa fluorescent cell line of interest. A fluorescent cell line of interestcan be grown in co-culture with one or more additional cell lines andthe growth of the fluorescent cell line of interest can be determined.For example, using the difference between the fluorescence signal of thefluorescent cell line of interest and a control indicates that the oneor more additional cell lines can be used to determine if the one ormore additional cell lines affects the growth rate of the fluorescentcell line of interest.

In some embodiments, the difference between the fluorescence signal(such as the intensity of the fluorescence signal at a particularwavelength, for example the emission maxima of the fluorescence signal)attributable to the fluorescent cell line of interest grown inco-culture with one or more additional cell lines relative to a controlis at least about 10%, meaning that the growth rate of the cell line ofinterest is either reduced or increased by at least about 10%, such asat least about 20%, at least about 30%, at least about 40%, at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 90%, at least about 100%, at least about 150%, at leastabout 200%, at least about 250%, at least about 300%, at least about350%, at least about 400%, at least about 500%, or greater then 500%. Insome embodiments, the difference is a statistically significantdifference. Thus, the presence of one or more additional cell lines caninduce a statistically significant difference in the growth rate of afluorescent cell line of interest, as compared to the control, such asvalue indicative of the basal rate of growth of the fluorescent cellline, or the fluorescent cell line of interest grown in the absence ofthe other cells or cell lines, for example grown in mono-culture. Insome examples, the additional cell line (or additional cell lines) willhave a negative impact on the first fluorescent cell line, such that thenumber of cells of the first fluorescent cell line is reduced as afunction of time relative to a control. In some examples, the additionalcell line (or additional cell lines) will have a positive impact on thefirst fluorescent cell line, such that the number of cells of the firstfluorescent cell line present in a cell culture increases as a functionof time relative to a control. It is also contemplated that thefluorescent cell line of interest can be co-cultured with primary cells,such as primary cells obtained from a subject, for example tumor cells,and the effect of the primary cells on the growth rate of thefluorescent cell line of interest determined.

In some embodiments, multiple fluorescent cell lines are grown inco-culture. Thus, the effect of each fluorescent cell line on the otherfluorescent cell line(s) present can be determined, for example in amultiplex assay. For example, using appropriate filters or FACS analysisamong other techniques, fluorescent cell lines expressing differentfluorescent proteins, such as red, green, yellow, cyan and the likefluorescent proteins can be discriminated and the fluorescent signalattributable to the different cell lines determined. Thus, the growthrates of individual fluorescent cell lines can be determined from amono-culture and/or a co-culture of two or more fluorescent cell lines.Such analysis greatly enhances the information that can be obtainedabout the individual fluorescent cell lines.

In addition to determining the effect of cell lines on a fluorescentcell line of interest, the growth assays can be used to determine if anexogenous agent, such as a test agent, for example a chemical agent,affects the growth of a fluorescent cell line of interest. This can alsobe extended to multiple cell lines (either fluorescent or not grown inco-culture, for example in a multiplex assay). In some embodiments, thedisclosed growth assay is used to determine if an exogenous agent, suchas a test agent (for example a potential modulator of angiogenesis, suchas a potential inhibitor of angiogenesis or a potential stimulator ofangiogenesis), growth factor, patient sample, etc. affects the growthrate of a fluorescent cell line of interest, such as one or more of thefluorescent cell lines disclosed herein. In addition, the differentialeffect of the exogenous agent on the different cell lines can bedetermined, as can the combinatorial effect of the exogenous agent andthe cells on a cell line of interest.

A fluorescent cell line of interest can be contacted with an exogenousagent and the impact of the exogenous agent on the growth of thefluorescent cell line of interest can be determined. For example, adifference between the fluorescence signal of the fluorescent cell lineof interest and a control indicates that the exogenous agent, such as atest agent (for example a potential modulator of angiogenesis, such as apotential inhibitor of angiogenesis or a potential stimulator ofangiogenesis), growth factor, patient sample, different cell line, etc.is a modulator (such as an inducer or inhibitor) of angiogenesis. Thus,in several embodiments, one or more of the disclosed fluorescent celllines growing in culture are contacted with a test agent (or testagents) to determine if the test agent is a modulator of angiogenesis.Exemplary test agents are given below. Following contact with theexogenous agent, the fluorescence of the culture can be measured versustime and/or concentration to determine the impact of the exogenous agenton the one or more fluorescent cell lines present in the culture. Forexample, the fluorescence signal generated by a fluorescent cell line ofinterest (such as the intensity of the fluorescence signal at aparticular wavelength, for example the emission maxima of thefluorescence signal) can be measured to determine if the fluorescencesignal attributable to the fluorescent cell line of interest (such asthe intensity of the fluorescence signal at a particular wavelength, forexample the emission maxima of the fluorescence signal) is increasing asa function of concentration of the exogenous agent, time, or both, forexample by comparison with a control, such as a value indicative of thebasal rate of growth of the fluorescent cell line of interest or thefluorescent cell line of interest not contacted with the exogenousagent. In several embodiments, the control is a known value indicativeof normal growth of the fluorescent cell line of interest, for examplethe doubling time of cellular number. In some embodiments, the controlis the fluorescence signal of a culture of cells (typically, but notnecessarily, a culture of the fluorescent cell line of interest) notcontacted with the exogenous agent.

In some embodiments, an exogenous agent, such as a test agent, decreasesthe growth rate of the fluorescent cell line of interest. A test agentexhibiting such an activity is identified as an inhibitor ofangiogenesis and would be of use in treating a disease or condition inwhich normal angiogenesis is increased, for example cancer. In someembodiments, a decrease in the growth rate of the fluorescent cell lineof interest relative to a control is at least about a 30%, at leastabout a 40%, at least about a 50%, at least about a 60%, at least abouta 70%, at least about a 80%, at least about a 90%, at least about a100%, at least about a 150%, at least about a 200%, at least about a250%, at least about a 300%, at least about a 350%, at least about a400%, at least about a 500% decrease. Because the fluorescence signalattributable to a fluorescent cell line of interest is proportional tothe number of cells of the cell line of interest present, the percentagedecrease can be measured as a percentage decrease in the fluorescentsignal, for example the fluorescence intensity at a particularwavelength, such as the emission maxima, attributable to the cell lineof interest. In additional embodiments, the decrease is a statisticallysignificant decrease as compared to a control.

In other embodiments, the exogenous agent, such as a test agent,increases the growth of the fluorescent cell line as compared to acontrol. A test agent exhibiting such an activity is identified as astimulator of angiogenesis and would be of use in treating a disease orcondition in which normal angiogenesis is inhibited. In someembodiments, an increase in the growth of the fluorescent cell line isat least about a 30%, at least about a 40%, at least about a 50%, atleast about a 60%, at least about a 70%, at least about a 80%, at leastabout a 90%, at least about a 100%, at least about a 150%, at leastabout a 200%, at least about a 250%, at least about a 300%, at leastabout a 350%, at least about a 400%, at least about a 500% increase ascompared to control. Because the fluorescence signal attributable to afluorescent cell line of interest is proportional to the number of cellsof the cell line of interest present, the percentage increase can bemeasured as a percentage increase in the fluorescent signal, for examplethe fluorescence intensity at a particular wavelength, such as theemission maxima, attributable to the cell line of interest. Inadditional embodiments, the increase is a statistically significantincrease as compared to a control.

ii. Tubule Formation Assay

Similarly to the growth assay, cultures of fluorescent cell linesexpressing different fluorescent proteins, such as the fluorescent celllines disclosed herein can be applied to tubule formation assays.Formation of new blood vessels is fundamental to angiogenesis and is thefocus of many drug screening and cell signaling studies. Blood vesseldevelopment is a significant event in the development and growth ofsolid tumors, and is involved in wound healing, retinopathy and maculardegeneration. The disclosed fluorescent cell lines, and in particularthe disclosed endothelial fluorescent cell lines are ideal for use inassays for assessing the degree of blood vessel formation using in vitrocell culture assays (see for example Auerbach et al. 2003. ClinicalChemistry 49:1, 32-40. 2. Taraboletti and Giavazzi, 2004 EJC. 40,881-889). Because no fluorescent/colorimetric staining is needed, thetubule formation assay can be followed over time and can be directlyvisualized used in existing instrumentation, such as the BD Pathway™Bioimager (BD Bioscience, San Jose, Calif.). This allows for the studyof the interaction between different cells types in this angiogenesis invitro assay. In addition, the tubule formation potential can also bedetermined for a co-culture of a fluorescent cell line of interest withprimary cells, such as primary cells obtained from a subject, forexample tumor cells.

Tubule Formation assays are typically based on the ability ofendothelial cells, such as the fluorescent endothelial cells disclosedherein, to form distinct blood-vessel-like tubules in an extracellularmatrix (such as BD Matrigel™ Matrix available from BD Bioscience, BMEavailable from Trevigen, or Geltrex™ available from Invitrogen®, and thelike). In other examples, tubule formation assays involve observation ofthe tubule forming potential of certain tumor cell lines (e.g.MDA-MB-435) and pericytes. The cells are visualized under microscopy,such as fluorescence microscopy in the case of the fluorescent celldisclosed herein, and the ability of a fluorescent cell line of interestto form tubules (also called the tubule formation potential) isdetermined. The determination of tubule formation can be performed bymanual tracing or by automated confocal imaging system, for exampleusing a BD Pathway™ Bioimager in conjunction with AngioApplication™.Using the disclosed fluorescent cell lines, tubule formation assays canbe performed on live cells, for example to avoid artifact that may arisefrom fixation artifacts, such as the disruption of tubules. Severalparameters can be measured in tubule formation assays, such as the totalarea of the tubules, the total number of tubules, number of nodes,number of branch points, the number of tubes per node, and/or node area.In some embodiments, the tubule formation potential is determined by acomputer implemented method, for example using the programAngioApplication™.

The fluorescent cell lines disclosed herein can be used to determine theeffects of an exogenous agent, such as cell lines and test agents, ontubule formation. In some embodiments, multiple fluorescent cell linesare grown in co-culture. Thus, the effect of each fluorescent cell lineon the other fluorescent cell line(s) present can be determined, or thedifferential effect of an exogenous agent, such as a test agent, orpatient sample, on the different cell lines can be assessed in amultiplex assay. For example using appropriate filters, the fluorescentsignal from fluorescent cell lines expressing different fluorescentproteins, such as red, green, yellow, cyan fluorescent proteins can bediscriminated and the fluorescent signal attributable from the differentfluorescent cell lines determined. Thus, the tubule formation potentialof individual cell lines can be determined from a mono-culture or even aco-culture, for example a co-culture of more than one fluorescent cellline.

When grown in co-culture, a difference between the tubule formationpotential of the fluorescent cell line of interest from a control, sucha mono-culture of the fluorescent cell line of interest indicates thatthe other cell line(s) is a modulator of angiogenesis, as evidenced bythe difference in tubule formation potential. In some embodiments, thedifference between the tubule formation potential, for example asmeasured by the number of least one of the total area of the tubules,the total number of tubules, number of nodes, number of branch points,the number of tubes per node, or node area formed in the co-culture ofthe fluorescent cell line of interest relative to a control is at leastabout 10%, such as at least about 20%, at least about 30%, at leastabout 40%, at least about 50%, at least about 60%, at least about 70%,at least about 80%, at least about 90%, at least about 100%, at leastabout 150%, at least about 200%, at least about 250%, at least about300%, at least about 350%, at least about 400%, at least about 500%, orgreater then 500%. In some embodiments, the difference is astatistically significant difference. Thus, a cell line can induce astatistically significant difference in the tubule formation potentialof a fluorescent cell line of interest, such as one of the disclosedfluorescent cell lines. Taking a combinatorial approach the impact ofmultiple different cell lines either alone or in combination on thetubule formation potential of the fluorescent cell line of interest canbe determined. In some examples, the presence of one or more additionalcell lines increases the tubule formation potential of the fluorescentcell line of interest, for example as measured by the total area of thetubules, the total number of tubules, number of nodes, number of branchpoints, the number of tubes per node, or node area formed by thefluorescent cell line of interest. These cell lines would be identifiedas positive regulators of angiogenesis. In some examples, the presenceof one or more additional cell lines decreases the tubule formationpotential of the fluorescent cell line of interest, for example asmeasured by the total area of the tubules, the total number of tubules,number of nodes, number of branch points, the number of tubes per node,or node area formed by the fluorescent cell line of interest. These celllines would be identified as negative regulators of angiogenesis.

Utilizing the disclosed fluorescent cell lines, tubule formation assayscan also be used to screen for a biological effect of a test agent, suchas the effect of potential modulators of angiogenesis. In someembodiments, a fluorescent cell line of interest (or multiple cell linesof interest in a multiplex assay) can be contacted with an exogenousagent, such as a cell line or test agent, and the impact of theexogenous agent on tubule formation potential can be determined.Exemplary test agents are given below. For example using the differencebetween the total area of the tubules, the total number of tubules,number of nodes, number of branch points, the number of tubes per node,and/or node area between a fluorescent cell line of interest and acontrol are used to determine if an exogenous agent, such as a testagent, impacts the ability of a fluorescent cell line of interest toform tubules. A difference between the tubule formation potential of afluorescent cell line of interest contacted with an exogenous agent anda control (such as a control culture exposed to the exogenous agent)indicates that the exogenous agent is a modulator of angiogenesis. Insome embodiments, the difference between the tubule formation potentialof the fluorescent cell line contacted with an exogenous agent relativeto a control is at least about 10%, such as at least about 20%, at leastabout 30%, at least about 40%, at least about 50%, at least about 60%,at least about 70%, at least about 80%, at least about 90%, at leastabout 100%, at least about 150%, at least about 200%, at least about250%, at least about 300%, at least about 350%, at least about 400%, atleast about 500%, or greater then 500%. In some embodiments, thedifference is a statistically significant difference. Thus, an exogenousagent can induce a statistically significant difference in the tubuleformation potential of the fluorescent cell line of interest contactedwith the test agent, as compared to the control, such as the fluorescentcell line of interest not contacted with the exogenous agent.

In one embodiment, the exogenous agent decreases ability of afluorescent cell line of interest to form tubules. A test agentexhibiting such an activity is identified as an inhibitor ofangiogenesis and would be of use in treating a disease or condition inwhich normal angiogenesis is increased, for example cancer. In someembodiments, a decrease in the tubule formation potential of thefluorescent cell line of interest is at least about a 30%, at leastabout a 40%, at least about a 50%, at least about a 60%, at least abouta 70%, at least about a 80%, at least about a 90%, at least about a100%, at least about a 150%, at least about a 200%, at least about a250%, at least about a 300%, at least about a 350%, at least about a400%, at least about a 500% decrease as compared to control. Inadditional embodiments, the decrease is a statistically significantdecrease as compared to a control.

In another embodiment, the exogenous agent increases the potential of afluorescent cell line of interest to form tubules as compared to acontrol, such as the fluorescent cell line of interest that has not beencontacted with the exogenous agent. A test agent exhibiting such anactivity is identified as a stimulator of angiogenesis and would be ofuse in treating a disease or condition in which normal angiogenesis isinhibited. In some embodiments, an increase in the growth of thefluorescent cell line is at least about a 30%, at least about a 40%, atleast about a 50%, at least about a 60%, at least about a 70%, at leastabout a 80%, at least about a 90%, at least about a 100%, at least abouta 150%, at least about a 200%, at least about a 250%, at least about a300%, at least about a 350%, at least about a 400%, at least about a500% increase as compared to control. In additional embodiments, theincrease is a statistically significant increase as compared to acontrol.

iii. Migration Assay

Another assay that can be used with the disclosed fluorescent cell linesis a cellular migration assay. These assays assess cellular migration ina controlled environment, such as a differential migration of the cellline, (or multiple cell lines in a multiplex assay) as determined byfluorescent signals (such as the intensity of a fluorescent signal of aparticular color, or at a particular wavelength, such as the emissionmaxima of a particular fluorescent protein) in a location that isassociated with migration to a particular location.

In one example, a cellular migration assay determines the ability ofcells to migrate up or down a chemical gradient. Migration “up” achemical gradient refers to migration from a region of lower chemicalconcentration of a chemical to a region of higher chemical concentration(for example migration toward a higher concentration of a chemicalattractant or away from a lower concentration of the chemicalattractant), while migration “down” a chemical gradient refers tomigration from a region of higher chemical concentration to a region oflower chemical concentration (for example migration away from a higherconcentration of a chemical repellent toward a lower concentration ofthe chemical repellent). Such migration is typically referred to aschemotaxis. Cells, such as the fluorescent cell lines disclosed herein,respond to chemical signals in their environment by the stimulation ofconcerted movement either toward a chemical attractant or away from achemical repellent. In mammalian cells, such as the fluorescent celllines disclosed herein, typical chemo-attractants include factorsexcreted by cells, for example factors found in serum, such as growthfactors and the like.

The disclosed fluorescent cells can be used in any cell migration assayformat, such as the ChemoTx™ system (NeuroProbe, Rockville, Md.)transwell system or any other suitable device or system. In someexamples, a cell migration assay is carried out as follows. A culture ofa fluorescent cell line of interest (such as any of the disclosedfluorescent cell lines or a mixture of such as fluorescent cell lines)is placed into a first chamber of a cell migration apparatus, and anexogenous agent (such as a chemoattractant) is placed in a secondchamber that is adjacent to and in communication with the first chamberof the cell migration apparatus, so that cellular migration from thefirst chamber to the second chamber can be detected. The chambers may beseparated by a membrane or filter that permits passage of cells from onechamber to the other chamber. The membrane or filter is configured suchthat the passive diffusion of the cells across the membrane or filter isminimized. In one example, the first chamber is the upper chamber of theapparatus and the second chamber is the lower chamber of the apparatus.In some examples the upper chamber is omitted and the cells are placeddirectly on a membrane or filter in communication with the lowerchamber. The ability of a fluorescent cell line such as the fluorescentcell lines disclosed herein to be stimulated to migrate can bedetermined. Typical migration assays have “unknown” sites (with cellsuspension above the filter and a solution containing the chemotacticfactor below it) and “negative control” sites (with cell suspensionabove the filter and suspension media, but no chemotactic factor,below). Random migration of unstimulated cells will account for some ofthe cells that pass through the filter. Migrated cells at the negativecontrol sites show the extent of unstimulated random migration, whichcan then be differentiated from chemotactic migration, or chemotaxis.Cells that stably express a fluorescent protein, such as the disclosedfluorescent cells can be read in a microplate with a fluorescencemicroplate reader. Thus, the number of fluorescent cells present ineither the upper chamber, lower chamber, or both chambers can bedetermined, for example as a function of time.

In some embodiments, the disclosed migration assay is used to determineif an exogenous agent affects or differentially affects the migration ofone or more of the fluorescent cell line of interest, such as one ormore of the fluorescent cell lines disclosed herein. A fluorescent cellline of interest can be contacted with exogenous agent and the impact ofthe exogenous agent on the migration of the fluorescent cell line ofinterest can be determined. For example, a difference between the numberof cells that migrate between a fluorescent cell line of interestcontacted with an exogenous agent and a control indicates that theexogenous agent, such as a test agent, cell line, growth factor, etc.,is a modulator of cellular migration. In other embodiments, differencesin migration among different cell lines in the migration assay providean indication of differential migration of the different cell lines inresponse to the exogenous agent. In some embodiments, the differencebetween the number of cells that migrate of the fluorescent cell linecontacted with an exogenous agent relative to a control, (for example asmeasured by the fluorescence intensity of a fluorescent protein stablyand constitutively expressed by the cells) is at least about 10%, suchas at least about 20%, at least about 30%, at least about 40%, at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 90%, at least about 100%, at least about 150%, at leastabout 200%, at least about 250%, at least about 300%, at least about350%, at least about 400%, at least about 500%, or greater then 500%. Insome embodiments, the difference is a statistically significantdifference. Thus, an exogenous agent can induce a statisticallysignificant difference in the migration of a fluorescent cell line ofinterest contacted with the exogenous agent, as compared to the control,such as the fluorescent cell line of interest not contacted with theexogenous agent or a different cell line that has been mixed with thecell line of interest.

In one embodiment, the exogenous agent decreases the ability of afluorescent cell line of interest to migrate. A test agent with such anactivity is identified as an inhibitor of angiogenesis and would be ofuse in treating a disease or condition in which normal angiogenesis isincreased, for example cancer. In some embodiments, a decrease inmigration of the fluorescent cell line of interest is at least about a30%, at least about a 40%, at least about a 50%, at least about a 60%,at least about a 70%, at least about a 80%, at least about a 90%, atleast about a 100%, at least about a 150%, at least about a 200%, atleast about a 250%, at least about a 300%, at least about a 350%, atleast about a 400%, at least about a 500% decrease as compared tocontrol. In additional embodiments, the decrease is a statisticallysignificant decrease as compared to a control.

In another embodiment, the exogenous agent increases the migration ofthe fluorescent cell line of interest as compared to a control. A testagent with such as activity is identified as a stimulator ofangiogenesis and would be of use in treating a disease or condition inwhich normal angiogenesis is inhibited. In some embodiments, an increasein migration of the fluorescent cell line is at least about a 30%, atleast about a 40%, at least about a 50%, at least about a 60%, at leastabout a 70%, at least about a 80%, at least about a 90%, at least abouta 100%, at least about a 150%, at least about a 200%, at least about a250%, at least about a 300%, at least about a 350%, at least about a400%, at least about a 500% increase as compared to control. Inadditional embodiments, the increase is a statistically significantincrease as compared to a control.

iv. Cell Viability Assay

Another example of an assay that can be used with the disclosedfluorescent cell lines is a cell viability assay. These assays are basedon the release of fluorescent protein from the cytoplasm of fluorescentcell lines that constitutively express fluorescent protein that occurswhen the integrity of the cell membrane of the cells is compromised, forexample when the cell dies, such as when the cell is exposed to acytotoxic agent, such as a test agent that is cytotoxic to the cell.Upon exposure to a cytotoxic agent the fluorescent protein is liberatedto the culture media and it can be measured, for example using afluorimeter. The greater the amount of fluorescent protein liberatedfrom the cells present in the culture, the greater the intensity of thefluorescence present in the media. The measured fluorescence in themedia corresponds to number of dead cells.

In some embodiments, the cell viability assay is used to determine if anexogenous agent is cytotoxic to one or more of the fluorescent celllines of interest, such as one or more of the fluorescent cell linesdisclosed herein. A fluorescent cell line of interest can be contactedwith exogenous agent and the impact of the exogenous agent on the deathof the fluorescent cell line of interest can be determined. For example,an increase in the relative florescence present in the media of betweena fluorescent cell line of interest contacted with an exogenous agentand a control indicates that the exogenous agent, such as a test agent,cell line, growth factor, etc., is cytotoxic to the cell line ofinterest. In other embodiments, differential cytotoxicity of anexogenous agent to different cell lines in the cell viability assayprovides an indication that a specific exogenous agent is preferentiallycytotoxic to one cell line but not other cell lines present in theculture. Such information is useful for screening agents that arepreferentially or differentially cytotoxic to a specific cell-type, forexample to the exclusion of other cell types. For example, in a mixedcell population a test agent could be screened to determine if it wascytotoxic (for example differentially cytotoxic) to diseased cells (suchas tumor cells) present in the mixed cell population, but not normalcells present in the mixed cell population.

In some embodiments, the difference between the fluorescence of themedia of a fluorescent cell line contacted with an exogenous agentrelative to a control, (for example as measured by the fluorescenceintensity of a fluorescent protein liberated from the cell line into themedia) is at least about 10%, such as at least about 20%, at least about30%, at least about 40%, at least about 50%, at least about 60%, atleast about 70%, at least about 80%, at least about 90%, at least about100%, at least about 150%, at least about 200%, at least about 250%, atleast about 300%, at least about 350%, at least about 400%, at leastabout 500%, or greater then 500%. In some embodiments, the difference isa statistically significant difference. Thus, an exogenous agent caninduce a statistically significant difference in the number of cellsthat die as a the migration of a fluorescent cell line of interestcontacted with the exogenous agent, as compared to the control, such asthe fluorescent cell line of interest not contacted with the exogenousagent or a different cell line that has been mixed with the cell line ofinterest.

The fluorescent cell lines of the present invention can be used in theabove-discussed assays to monitor endothelial cell responses to variousexogenous agents, including test agents. However, as described herein,endothelial cell responses to tumor cells in two-dimensional cultureassays are not correlative with observations of tumor-stimulatedangiogenesis in the in vivo whole animal context. In order to recreatethe in vivo tumor microenvironment in an in vivo context, athree-dimensional co-culture assay was developed, as discussed in detailbelow.

B. Immortalized Fluorescent Cell lines

Disclosed herein are immortalized mammalian cell lines that stablyexpress a fluorescent protein. The disclosed fluorescent cell lines areproduced in disclosed examples by transfecting mammalian expressionvectors for fluorescent proteins, such as green, yellow, red and bluefluorescent proteins and the like into a variety of cell lines such ascell lines derived from both vascular and lymphatic endothelial cells aswell as inflammatory cells (such as monocytes and mast cells) and tumorcells (such as tumor cells from lung, breast, and the like). Thetransfected cells are selected for stable (through antibioticresistance) and high-homogeneous expression (through flow cytometry cellsorting) of the fluorescent proteins.

In some embodiments, the disclosed mammalian cell line is stablytransfected with a mammalian expression vector that includes anucleotide sequence encoding the amino acid sequence of a fluorescentprotein, operably connected to a constitutively active promoter thatdrives the expression of the fluorescent protein and a nucleotidesequence encoding a selection marker. The disclosed fluorescent celllines stably effect high level expression of the fluorescent protein inthe absence of a selection agent and maintain high level expression ofthe fluorescent protein when the fluorescent cell lines proliferatethrough multiple passages, for example 10 passages, 20 passages, 30passages, 40 passages, 50 passages, 100 passages, 150 passages, 200passages, 250 passages, 300 passages, 400, or even greater than 500passages of the cell line.

The disclosed cell lines can be derived from any mammalian species, forexample humans, apes, monkeys, swine, bovine, and the like. In someembodiments, the fluorescent cell line is an endothelial cell line, forexample the porcine aortic endothelial cell line PAE (see for exampleFIG. 1A-1D), the human lymphatic endothelial cell line HMEC-1, or therhesus macaque choroidal endothelial cell line RF/6A (ATCC CRL-1780). Insome embodiments, the fluorescent cell line is an epithelial cell line,for example the human adenocarcinoma cell line A549. In someembodiments, the fluorescent cell line is an adenocarcinoma cell line,for example the human breast adenocarcinoma cell line MCF7. In someembodiments, the fluorescent cell line is a mast cell line, such as thehuman mast cell line HMC-1.

The fluorescent cell lines can be transfected with vectors expressingdifferent fluorescent proteins, such as green, yellow, red and cyanamong others, such that a cell line can be constructed that stably andconstitutively expresses each of the fluorescent proteins. In otherwords, a particular parental cell line can be divided into sub celllines, in which each of the sub cell lines expresses a differentfluorescent protein. The nucleic acids encoding fluorescent proteins canbe expressed in mammalian cell lines. Transfection of mammalian celllines with recombinant DNA may be carried out by conventional techniquesas are well known to those skilled in the art, for example as calciumphosphate coprecipitates, the use of conventional mechanical proceduressuch as microinjection, electroporation (for example using aNUCLEOFECTOR™ II available from AMAXA®) and insertion of a plasmidencased in liposomes.

Polynucleotide sequences encoding the fluorescent proteins can beoperatively connected to expression control sequences. An expressioncontrol sequence operatively connected to a coding sequence is ligatedsuch that expression of the coding sequence is achieved under conditionscompatible with the expression control sequences. The expression controlsequences include, but are not limited to appropriate promoters,enhancers, transcription terminators, a start codon (i.e., ATG) in frontof a protein-encoding gene, splicing signal for introns, maintenance ofthe correct reading frame of that gene to permit proper translation ofmRNA, and stop codons.

The polynucleotide sequences encoding the fluorescent proteins can beinserted into an expression vector including, but not limited to aplasmid, to allow insertion or incorporation of sequences into mammaliancell lines. Biologically functional plasmid DNA vectors capable ofexpression and replication in a mammalian cell line are known in theart. Examples of vectors that can be used in constructing the disclosedfluorescent cell lines include those vectors available from AMAXA®, suchas pmaxFP-Green-C, pmaxFP-Green-N, pmaxFP-Yellow-C, pmaxFP-Yellow-N,pmaxFP-Yellow-PRL, pmaxFP-Red-C, and pmaxFP-Red-N, and vectors availablefrom Clontech, such as pAcGFP1-Hyg-N1, pAcGFP1-N1, pAcGFP1-N2,pAcGFP1-N3, pAmCyan1-N1, pAsRed2-N1, pDsRed2-N1, pDsRed-Express-N1, pDsRed-Monomer-Hyg-N1, pDsRed-Monomer-N1, pHcRedl-N1/1, pZsGreenl-N1,pZsYellowl-N1 and the like.

Those of skill in the art will also recognize that the selection markercomponent of the vector need not be restricted to an antibioticresistance gene. By “selection marker” it is meant a gene encoding aprotein wherein an activity of the expressed protein is suitable forexerting selection pressure on the cell in which it is expressed. Manyselection markers are known to those of skill in the art, including butnot limited to resistance markers for antibiotics such as ampicillin,streptomycin, kanamycin, neomycin and the like. Any suitable selectionmarker may be utilized to construct the vectors of the presentdisclosure for transfection of the mammalian cell lines, so long as theselection marker provides appropriate selection pressure on the cellswithin which it is contained.

The disclosed cell lines can be further sorted by fluoresce activatedcell sorting (FACS) to select for cells from a particular cell line thathave the greatest fluorescence intensity for a particular fluorescentprotein, for example, by gating on the brightest population of cells andsorting these cells for further propagation. In certain embodiments, thedisclosed fluorescent cell lines have been sorted by FACS to select forcells that stably and constitutively express fluorescent proteins. Insome situations it is advantageous to FACS sort a florescent cell linemultiple times, such as 2, 3, 4, 5, 6, 7, 8, 9, or even greater than 9times to enrich for a population of cells that stably and constitutivelyexpresses a fluorescent protein.

The ability to create multiple different cell lines expressing differentfluorescent proteins enhances the ability to study cells in co-cultureswhere different cell lines are included in the same assay. Differentcombinations of fluorescent cells enable the study of the interactionbetween different cell types at the qualitative (morphological) andquantitative level. The use of these cells will also ease thedevelopment of new in vitro multicellular angiogenesis models, such asthose disclosed herein.

In particular, the disclosed cell lines are useful in the estimation ofthe angiogenic potential of patient serum samples and assessment ofphysiologically active angiogenic/antiangiogenic drug levels in patientsamples. The disclosed cell lines also are well suited for integrationinto existing angiogenesis assays, such as growth assays,migration/invasion assays, tubule formation assays, cell viabilityassay, cell to cell interaction assays, cell to matrix interactionassays, apoptosis assays, and are particularly amenable to study byfluorescent/confocal microscopy, for example to determine on a cell bycell basis the effects co-culture has on different cell lines, such ascell lines from different anatomical origins.

The disclosed cell lines can be integrated into existing kits to replacestandard reagents. Tubule formation assays are an example of an assaythat would substantially benefit from inclusion of one or more of thedisclosed cell lines. Typical tubule formation assays require thestaining of the cells with calcein AM prior to the assay. Multipleproblems are associated with this is approach, including interaction ofthe staining chemical with the cellular objects of study (calcein AM isa known inhibitor of certain cell types), inter-experimental variabilityof the staining protocol, and the inability to use different cell typeson the same assay. The use of the disclosed stably transfectedfluorescent cell lines in tubule formation assays would eliminate thestaining of cells with calcein, eliminate the variability in emittedfluorescence and greatly expand the capabilities of the assayintroducing the possibility of the use of multiple cell types in thesame assay.

Another advantage of the use of stable fluorescent cellular in vitroassays is that at any time during the assay the cells can be observedunder a fluorescent microscope, which allows for morphological analysisand comparison of mono-cultures versus co-cultures. For example, tubuleformation and morphological analysis is assessed as a function of time.

C. AngioApplicaton™

Angiogenesis in vitro assays (such as the tubule formation assaydisclosed herein) and ex vivo assays (such as chicken chorioallantoicassay) are fundamental tools to the angiogenesis field. One of thehurdles in determining the effects of exogenous agents in such assays,for example, the tubule formation assays disclosed herein, isdetermining and quantifying the effects of such exogenous agents on thetubule formation potential of the cell line or cell lines under study,for example the fluorescent cell lines disclosed herein. Automatedtechnologies assist with the precise morphological quantification of avasculature formation assay, such as the tubule formation assaydisclosed herein, for example to determine the total area of thetubules, the total number of tubules, number of nodes, number of branchpoints, the number of tubes per node, and/or node area in a tubuleformation assay. In one example, AngioApplication™ is a computerimplemented automated analysis used to analyze the morphology of thecell lines. AngioApplication™ is an image-analysis software thatautomatically quantifies morphological parameters in assays involvingformation of vasculature. The program reports the total area of thetubules, the total number of tubules, number of nodes, number of branchpoints, the number of tubes per node, or node among other parameters.This software utilizes the freely available NIH ImageJ library (Rasband,W. S., ImageJ, U.S. National Institutes of Health, Bethesda, Md., USA,available on the world wide web at rsb.info.nih.gov/ij; Abramoff et al.,Image Processing with ImageJ, Biophotonics International 11:7, 36-42,2004). As the program is coded in Java it can potentially be used in anycomputer platform (for example Windows, Macintosh, Unix, Linux, etc.).This program allows for the rapid/automated quantification ofangiogenesis assays thus enhancing the capabilities of existingtechnologies for robust drug screening, patient diagnosis, andassessment of biologically active angiogenic or antiangiogenic drugs inpatient samples.

As shown in FIG. 15, the AngioApplication™ graphical user interface(GUI) which allows the user to choose the image (or batch of images) tobe analyzed. The settings window of AngioApplication™ (see FIG. 16)allows the user to dynamically adjust several parameters to allow for amore precise analysis of the morphological features of the image, forexample total length of the tubules, the total area of the tubules, thetotal number of tubules, number of nodes, number of branch points, thenumber of tubes per node, and/or node area. The program has beendesigned to find tubes and nodes in the original image and produce an“overlay” which shows both structures colored differently (see forexample FIG. 17A and FIG. 17B). As shown in FIG. 17A and FIG. 17B bothfluorescent images (FIG. 17A) and bright field images (FIG. 17B) can beanalyzed. Images can be stored for later analysis (see FIG. 18) and thedata generated by AngioApplication™ are stored directly into an Excelfile (see FIG. 19).

D. Exemplary Test Agents

The methods disclosed herein are of use for identifying test agents thatare modulators of angiogenesis. A “test agent” is any substance or anycombination of substances that is useful for achieving an end or result.The test agents identified using the methods disclosed herein can be ofuse for affecting the normal angiogenic potential of a fluorescent cellline. Any test agent that has potential (whether or not ultimatelyrealized) to affect the angiogenic potential of the fluorescent celllines disclosed herein can be tested using the methods of thisdisclosure.

Exemplary test agents include, but are not limited to, peptides such as,soluble peptides, including but not limited to members of random peptidelibraries (see for example, Lam et al., Nature, 354:82-84, 1991;Houghten et al., Nature, 354:84-86, 1991), and combinatorialchemistry-derived molecular library made of D- and/or L-configurationamino acids, phosphopeptides (including, but not limited to, members ofrandom or partially degenerate, directed phosphopeptide libraries; forexample, Songyang et al., Cell, 72:767-778, 1993), antibodies(including, but not limited to, polyclonal, monoclonal, humanized,anti-idiotypic, chimeric or single chain antibodies, and Fab, F(ab′)₂and Fab expression library fragments, and epitope-binding fragmentsthereof), small organic or inorganic molecules (such as, so-callednatural products or members of chemical combinatorial libraries),molecular complexes (such as protein complexes), or nucleic acids.

Appropriate tests agents can be contained in libraries, for example,synthetic or natural compounds in a combinatorial library. Numerouslibraries are commercially available or can be readily produced; meansfor random and directed synthesis of a wide variety of organic compoundsand biomolecules, including expression of randomized oligonucleotides,such as antisense oligonucleotides and oligopeptides, also are known.Alternatively, libraries of natural compounds in the form of bacterial,fungal, plant and animal extracts are available or can be readilyproduced. Additionally, natural or synthetically produced libraries andcompounds are readily modified through conventional chemical, physicaland biochemical means, and may be used to produce combinatoriallibraries. Such libraries are useful for the screening of a large numberof different compounds.

Libraries (such as combinatorial chemical libraries) useful in thedisclosed methods include, but are not limited to, peptide libraries(for example see U.S. Pat. No. 5,010,175; Furka, Int. J. Pept. Prot.Res., 37:487-493, 1991; Houghton et al., Nature, 354:84-88, 1991; andPCT Publication No. WO 91/19735), encoded peptides (see for example PCTPublication WO 93/20242), random bio-oligomers (see for example PCTPublication No. WO 92/00091), benzodiazepines (see for example U.S. Pat.No. 5,288,514), diversomers such as hydantoins, benzodiazepines anddipeptides (see for example Hobbs et al., Proc. Natl. Acad. Sci. USA,90:6909-6913, 1993), vinylogous polypeptides (see for example Hagiharaet al., J. Am. Chem. Soc., 114:6568, 1992), nonpeptidal peptidomimeticswith glucose scaffolding (see for example Hirschmann et al., J. Am.Chem. Soc., 114:9217-9218, 1992), analogous organic syntheses of smallcompound libraries (see for example Chen et al., J. Am. Chem. Soc.,116:2661, 1994), oligocarbamates (see for example Cho et al., Science,261:1303, 1003), and/or peptidyl phosphonates (see for example Campbellet al., J. Org. Chem., 59:658, 1994), nucleic acid libraries (seeSambrook et al. Molecular Cloning, A Laboratory Manual, Cold SpringsHarbor Press, N.Y., 1989; Ausubel et al., Current Protocols in MolecularBiology, Green Publishing Associates and Wiley Interscience, N.Y.,1989), peptide nucleic acid libraries (see for example U.S. Pat. No.5,539,083), antibody libraries (see for example Vaughn et al., Nat.Biotechnol., 14:309-314, 1996; PCT App. No. PCT/US96/10287),carbohydrate libraries (see for example Liang et al., Science,274:1520-1522, 1996; U.S. Pat. No. 5,593,853), small organic moleculelibraries (see for example benzodiazepines, Baum, C&EN, Jan 18, page 33,1993; isoprenoids, U.S. Pat. No. 5,569,588; thiazolidionones andmethathiazones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat. Nos.5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. No. 5,506,337;benzodiazepines, U.S. Pat. No. 5,288,514) and the like.

Libraries useful for the disclosed screening methods can be produced ina variety of manners including, but not limited to, spatially arrayedmultipin peptide synthesis (see for example Geysen, et al., Proc. Natl.Acad. Sci., 81(13):3998 4002, 1984), “tea bag” peptide synthesis (seefor example Houghten, Proc. Natl. Acad. Sci., 82(15):51315135, 1985),phage display (see for example Scott and Smith, Science, 249:386-390,1990), spot or disc synthesis (see for example Dittrich et al., Bioorg.Med. Chem. Lett., 8(17):23512356, 1998), or split and mix solid phasesynthesis on beads (see for example Furka et al., Int. J. Pept. ProteinRes., 37(6):487 493, 1991; Lam et al., Chem. Rev., 97(2):411-448, 1997).Libraries may include a varying number of compositions (members), suchas up to about 100 members, such as up to about 1000 members, such as upto about 5000 members, such as up to about 10,000 members, such as up toabout 100,000 members, such as up to about 500,000 members, or even morethan 500,000 members.

In one convenient embodiment, high throughput screening methods involveproviding a combinatorial chemical or peptide library containing a largenumber of potential therapeutic compounds. Such combinatorial librariesare then screened in one or more assays as described herein to identifythose library members (particularly chemical species or subclasses) thatdisplay a desired characteristic activity (such as in an increase ordecrease in tubule formation). In one example a test agent of use isidentified that increases the number of tubules formed. In anotherexample a test agent of use is identified that inhibits tubuleformation, for example by decreasing the relative number of tubulesformed.

The compounds identified using the methods disclosed herein can serve asconventional “lead compounds” or can themselves be used as potential oractual therapeutics. In some instances, pools of candidate agents may beidentify and further screened to determine which individual or subpoolsof agents in the collective have a desired activity.

E. Therapeutic Compounds, Formulations and Treatments

This disclosure further relates to methods for modulating angiogenesisin a subject. The disclosed methods can identify compounds that modulateangiogenesis. The compounds and derivatives thereof are particularlyuseful for modulating angiogenesis in a subject, such as a subjectsuffering from a disease or condition accompanied by deregulatedangiogenesis, for example cancer. The methods of modulating angiogenesisinclude administering to a subject a therapeutically effective amount ofa test agent identified as one that modulates angiogenesis. Thus in someembodiments, the pharmaceutical compositions containing a test agentthat decreases angiogenesis is administered to a subject, such as asubject with cancer. In some embodiments, the subject is a humansubject. It is also contemplated that the compositions can beadministered with conventional treatments for cancer, such as inconjunction with a therapeutically effective amount chemotherapeuticagent.

In some examples, a subject is selected for treatment with anangiogenesis modulator that increases angiogenesis. Such a subject canbe treated with a test agent identified by the methods disclosed hereinthat increase angiogenesis. In some embodiments, the subject is a humansubject.

Therapeutic compound(s) can be administered directly to a subject forexample a human subject. Administration is by any of the routes normallyused for introducing a compound into ultimate contact with the tissue tobe treated. The compounds are administered in any suitable manner,optionally with pharmaceutically acceptable carrier(s). Suitable methodsof administering therapeutic compounds are available and well known tothose of skill in the art, and although more than one route can be usedto administer a particular composition, a particular route can oftenprovide a more immediate and more effective reaction than another route.

When the test agent is to be used as a pharmaceutical, the test agent isplaced in a form suitable for therapeutic administration. The test agentmay, for example, be included in a pharmaceutically acceptable carriersuch as excipients and additives or auxiliaries, and administered to asubject. Frequently used carriers or auxiliaries include magnesiumcarbonate, titanium dioxide, lactose, mannitol and other sugars, talc,milk protein, gelatin, starch, vitamins, cellulose and its derivatives,animal and vegetable oils, polyethylene glycols and solvents, such assterile water, alcohols, glycerol and polyhydric alcohols. Intravenousvehicles include fluid and nutrient replenishers. Preservatives includeantimicrobial, anti-oxidants, chelating agents and inert gases. Otherpharmaceutically acceptable carriers include aqueous solutions, nontoxicexcipients, including salts, preservatives, buffers and the like, asdescribed, for instance, in Remington's Pharmaceutical Sciences, 15thed., Easton: Mack Publishing Co., 1405-1412, 1461-1487, 1975, and TheNational Formulary XIV., 14th ed., Washington: American PharmaceuticalAssociation, 1975). The pH and exact concentration of the variouscomponents of the pharmaceutical composition are adjusted according toroutine skills in the art. See Goodman and Gilman The PharmacologicalBasis for Therapeutics, 7th ed.

The pharmaceutical compositions are in general administered topically,intravenously, orally or parenterally or as implants. Suitable solid orliquid pharmaceutical preparation forms are, for example, granules,powders, tablets, coated tablets, (micro)capsules, suppositories,syrups, emulsions, suspensions, creams, aerosols, drops or injectablesolution in ampoule form and also preparations with protracted releaseof active compounds, in whose preparation excipients and additivesand/or auxiliaries such as disintegrants, binders, coating agents,swelling agents, lubricants, flavorings, sweeteners or solubilizers arecustomarily used as described above. The pharmaceutical compositions aresuitable for use in a variety of drug delivery systems. For a briefreview of methods for drug delivery, see Langer, Science, 249:1527-1533,1990, which is incorporated herein by reference.

For treatment of a patient, depending on activity of the compound,manner of administration, nature and severity of the disorder, age andbody weight of the patient, different daily doses are necessary. Undercertain circumstances, however, higher or lower daily doses may beappropriate. The administration of the daily dose can be carried outboth by single administration in the form of an individual dose unit orelse several smaller dose units, and also by multiple administrations ofsubdivided doses at specific intervals.

A therapeutically effective dose is the quantity of a compound accordingto the disclosure necessary to prevent, to cure or at least partiallyameliorate the symptoms of a disease and its complications. Amountseffective for this use will, of course, depend on the severity of thedisease and the weight and general state of the patient. Typically,dosages used in vitro may provide useful guidance in the amounts usefulfor in situ administration of the pharmaceutical composition, and animalmodels may be used to determine effective dosages for treatment ofparticular disorders. Various considerations are described, e.g., inGilman et al., eds., Goodman and Gilman: the Pharmacological Bases ofTherapeutics, 8th ed., Pergamon Press, 1990; and Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa.,1990. Effectiveness of the dosage can be monitored by any method.

F. Detection

The disclosed fluorescent cell lines can be detected by detecting thepresence of the emission spectrum of the fluorescent proteins expressedby the fluorescent cell lines, for example using appropriate filtersand/or monochrometers, (for excitation, emission, or both) the differentfluorescent proteins can be detected using fluorescence microscopy andby FACS. However, it will be readily understood by those of skill in theart that other means for detecting the presence of fluorescent proteinsand thus the cells expressing such protein may also be used.

Separate populations of fluorescent proteins with different emissionspectra can be used to identify the cells containing such proteins, suchas the disclosed fluorescent cell lines. For example, the characteristicemissions from the different fluorescent proteins can be observed ascolors or can be decoded to provide information about the particularwavelength at which the emission is observed, for example to identifythe number of cells of a particular kind or the location of such cell.Methods and devices for eliciting and detecting emissions fromfluorescent proteins are well known in the art. In brief, a light sourcetypically has a range that emits light at a wavelength shorter than thewavelength to be detected is used to elicit an emission by thefluorescent proteins. Numerous such light sources (and devicesincorporating such light sources) are known in the art, includingwithout limitation: deuterium lamps and xenon lamps equipped withfilters, continuous or tunable gas lasers, such as argon ion, HeCdlasers, solid state diode lasers (for example, GaN, GaAs lasers), YAGand YLF lasers and pulsed lasers. The emissions of fluorescent proteinscan similarly be detected using known devices and methods, includingwithout limitation, spectral imaging systems. Optionally, the emissionsare passed through one or more filters or prisms prior to detection. Thesimultaneous multicolor wavelength, such as multicolor, identificationof fluorescent proteins permits rapid identification of cell withoutrequiring fixation of the cells.

G. Kits and High Throughput Systems

This disclosure also provides kits including one or more of thefluorescent cell lines disclosed herein. Such kits can be used for thestudy of angiogenesis, for example to identify test agents that modulateangiogenesis, or the impact of mixed populations of cell types onangiogenic potential. The kits include at least one of the fluorescentcell lines disclosed herein. The kits may further include additionalcomponents such as instructional materials and additional reagents (forexample serum, growth media and the like). The kits may also includeadditional components to facilitate the particular application for whichthe kit is designed (for example microtiter plates, optical filters andthe like). Such kits and appropriate contents are well known to those ofskill in the art. The instructional materials may be written, in anelectronic form (such as a computer diskette or compact disk) or may bevisual (such as video files). It is contemplated that the kits cancontain reagents for carrying out the assays described herein, forexample reagents for migration, proliferation, and/or tubule formationassays. In some examples, the kit also includes the AngioApplication™program, for example supplied on a digital medium (such as a computerdiskette or compact disk).

This disclosure also provides integrated systems for high-throughputscreening of test agents for modulation of angiogenesis. The systemstypically include a robotic armature that transfers fluid from a sourceto a destination, a controller that controls the robotic armature, a tagdetector, a data storage unit that records tag detection, and an assaycomponent such as a microtiter dish comprising a well having a cellculture, for example a cell culture containing one or more of thefluorescent cell lines disclosed herein.

A number of robotic fluid transfer systems are available, or can easilybe made from existing components. For example, a Zymate XP (ZymarkCorporation; Hopkinton, Mass.) automated robot using a Microlab 2200(Hamilton; Reno, Nev.) pipetting station can be used to transferparallel samples to 96 well microtiter plates to set up several parallelsimultaneous assays of fluorescent cell lines, for example the assay theeffect of one or more test agents on angiogenesis.

Optical images can viewed (and, if desired, recorded for futureanalysis) by a camera or other recording device (for example, aphotodiode and data storage device) and are optionally further processedsuch as by digitalizing, storing, and analyzing the image on a computer.A variety of commercially available peripheral equipment and software isavailable for digitizing, storing and analyzing a digitized video ordigitized optical image, for example, using PC (Intelx86 or Pentiumchip-compatible DOS™, OS2™ WINDOWS™, WINDOWS NT™ or WINDOWS95™ basedcomputers), MACINTOSH™, or UNIX based (for example, a SUN™, a SGI™, orother work station) computers.

H. Three-Dimensional Co-Cultures

The stably-transfected fluorescent cells provided herein can be used tomonitor the response of endothelial cells in a co-culture to one or moretest agents, such as pharmaceutical agents. The detection of endothelialcell proliferation, motility and tubule formation in response to one ormore test agents indicates the angiogenic effect of the test agent andcan provide crucial information related to development of patienttherapies. However, as described herein, endothelial cell responses totumor cells co-cultured in two-dimensions (for example, separated by alayer of gel matrix such as a BME matrix) do not correlate with the invivo angiogenic tumor activity.

To overcome deficiencies of 2D in vitro co-cultures, described hereinare three-dimensional (3D) in vitro co-cultures that provide a mimeticof in vivo tumor activity. The co-cultures can be prepared in anysuitable culture vessel or chamber, including multi-well ormulti-chamber culture plates.

The 3D co-cultures described herein are prepared in three layers, whichcan be of any volume or thickness. The first layer, which is in contactwith the bottom of the culture vessel, is any solidified polymer ofneutral charge that is known to the art, and which does not alter thebiological activity of the cells in culture. In particular examples, thepolymer is a polysaccharide of neutral charge. In particular examples,the first layer comprises solidified agarose. In other examples, thislayer comprises a neutral polysaccharide polymer of cellulose, curdlan,cellulose, starch, glycogen, chitin, and the like.

The second layer of the 3D co-cultures comprises a mixture of two ormore types of cells embedded in an extracellular matrix gel extract orany suitable synthetic gel product. Exemplary extracellular matrix gelsinclude BD MATRIGEL™ gel matrix (BD Bioscience), BME (Trevigen),GELTREX® gel matrix (Invitrogen), Collagen Type I/IV and the like. Thegel matrix layer also comprises a mixture of endothelial cells that aredispersed (distributed) throughout the second layer and tumor cells. Inparticular examples, the endothelial cells can be any immortalizedendothelial cell line, including the stably-transfected fluorescentendothelial cells described herein. In other examples, the endothelialcells are non-immortalized endothelial cell cultures that aretransiently transfected with a fluorescent protein. The tumor cells canbe derived from a cell line or from a tumor biopsy extracted from asubject. In particular embodiments, the tumor cells are in the form of atumor spheroid colony. In other embodiments, the tumor cells are a pieceof a tumor biopsy. The tumor cells can also be stably transfected toexpress a fluorescent protein.

In other examples, the second layer includes one or more additionalmammalian cell types. Any cell type known to the art that is or might bepart of the tumor microenvironment can be included in the gel matrixlayer, for example, macrophages, mast cells, fibroblasts, adipocytes,and pericytes can all (independently or in combination) be included inthe co-culture. In particular examples, multiple cell lines included inthe second layer are transfected with constructs that expressfluorescent proteins of distinguishable emission spectra.

The third layer of the 3D co-cultures is comprised of any suitableliquid culture medium, and is provided on top of the second layer. Anymammalian tissue culture media known to the art can be used; thus, askilled artisan will understand the parameters that will influenceselection and adaptation of media for cell growth.

In particular embodiments, one or more test agents, such as thosedescribed herein, is added to any layer of the co-culture, such as thefirst, second, or third layer of the co-culture. In particular examples,the test agent is added to the third layer of the co-culture. Inparticular examples, the test agent is one or more anti-angiogenicand/or anti-metastatic compound. In other examples, the test agent is apromoter of angiogenesis or metastasis. In some examples, the test agentdirectly affects angiogenesis, in the manner of drugs such as Avastin®.In other examples the test agent is one or more indirect anti-angiogeniccompounds such as “non steroidal anti-inflammatory drugs” or NSAIDs.

In particular embodiments, the 3D co-cultures described herein are usedto monitor the angiogenic or metastatic potential of tumor cells. Suchmethods involve preparing a 3D co-culture (in three layers, as describedherein), incubating the co-culture for a period of time, and detectingendothelial cell proliferation or tubule formation, or angiotropism (forinstance, using the assays described herein). In particular examples,the tumor cells are derived from a tumor removed from a subject and the3D co-cultures can be used to assess the angiogenic or metastaticpotential of the tumor. The co-culture can be incubated for any lengthof time necessary to observe the angiogenic or metastatic activityincluding incubation times from several hours (6, 7, 8, 9 or more hours)to several days (1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days) to one, twoor more weeks. The detection of endothelial cell proliferation, tubuleformation, or angiotropism is achieved by fluorescence or standardmicroscopy alone or confocal laser scanning microscopy or in combinationwith the detection methods described herein. In particular embodiments,the methods of monitoring angiogenic or metastatic potential of tumorcells are used to determine the effects of at least one test agent onthe angiogenic or metastatic potential of tumor cells derived from asubject. In such examples, the test agent may be a candidate therapeuticcompound that may be used for treatment of the subject. In otherexamples, it may be a compound that has already been administered to asubject (either the same subject or a subject different from the subjectfrom whom the tumor cells originated) as part of a cancer treatmentregimen. In this way, the development of drug-resistance of a tumor andthe efficacy of a treatment in a subject can be monitored by repeatedlyusing the 3D co-cultures over a period of time.

The development of 3D co-cultures that provide a mimetic of in vivotumor activity also enables methods for selecting personalizedanti-angiogenic and anti-metastatic therapies (that is, therapies thatare selected in order to be specifically effective in a specificsubject). These methods involve assaying the anti-angiogenic oranti-metastatic activity of a panel of compounds or other treatmentvariables (e.g., dosage, timing, etc.), alone or in combination, using a3D co-culture that contains tumor cells from the specific subject. Suchassays optionally can be done in a multi-well plate. 3D co-cultures areprepared as described using the target subject's tumor cells, and atleast one test compound (test agent) is added to the third layer of eachof some but not all of the 3D co-cultures. The co-cultures are incubatedand angiogenic or metastatic activity is observed, for instance asdescribed herein. The test agent (or dosage, or other variable regimen)that has the most beneficial (strongest) anti-angiogenic and/oranti-metastatic effect, for instance in comparison to the angiogenic ormetastatic activity observed in the compound-free co-culture, is thenselected for personalized treatment. The continued efficacy of suchtherapies can be monitored after any desired length of time, such asthree months or longer if necessary.

In another embodiment, the 3D co-cultures can also be used in thedevelopment of computer programs to quantitate angiogenesis and vesselcomplexity in three-dimensional projections. Such programs wouldultimately be applied to analyze the vascular network around tumors anddetermine a 3D vascular density profile for a given tumor via CAT or MRIscans. Such images could be retaken after initial anti-angiogenic drugtherapy to quantitate treatment regimen efficacy before actually seeingalterations in tumor size.

EXAMPLES Example 1 Generation of Stably Transfected Fluorescent Cells

This example describes the materials and methods used in the generationof stably transfected fluorescent cells.

Cell Lines.

Cell lines A549 (lung adenocarcinoma) and MCF-7 (breast cancer) wereacquired though the DTP 60 cell line library at NCI/Frederick. RF/6A(ATCC CRL-1780) was obtained from the American Type Culture Collectionas a Macaca mulatta (rhesus monkey) retina endothelial cell line butshown to be a monkey pericyte cell line via surface marker expression.The mast cell line HMC-1 was derived from primary mast cells exposed to5-azacytindine, spontaneously immortalized and established by Dr. JohnButterfield (Butterfield et al., Leuk. Res. 12:345-355, 1988) and wasobtained from the Department of Internal Medicine, Division of AllergicDiseases, Mayo Clinic, Rochester Minn. 55905. Endothelial cell lineHMEC-1 is a human dermal microvascular blood vessel endothelial cellline originally developed by Dr. Thomas Lawley (Emory University Schoolof Medicine, Atlanta, Ga., USA) via SV40 large T transfection (Ades etal., J. Invest. Dermatol. 99:683-699, 1992), and was obtained from Dr.Hynda Kleinman (NIDCR). PAE is a porcine aortic endothelial cell linefrom Dr. Carl-Henrik Heldin (Ludwig Institute for Cancer Research,Uppsala, Sweden) that became spontaneously immortalized with continuouspassaging (Ronnstrand et al. EMBO J. 11:3911-3919, 1992).

Plasmids.

Plasmids pmaxFP-GFP-C, pmaxFP-Yellow-C and pmax-Red-N were obtained fromAMAXA® Inc. (Gaithersburg, Md.). Plasmids pDsRed2-C1 and pAmCyan1-C1were obtained from BD Bioscience. pcDNA3.1-GFP was built in houseinserting the GFP coding reading frame into the pcDNA3.1-TOPO-TA(INVITROGEN™) backbone. All plasmids contain a GENETICIN® selectablemarker.

Transfections and Generation of Stable Transfectants.

Cell lines A549, MCF7, SK-LMS-1, 92-1, PC-12, HMEC-1, RF/6A (ATCCCRL-1780), PAE, and HMC-1 were stably transfected with the plasmidsdescribed above using a NUCLEOFECTOR™ II (AMAXA® Inc.). Differenttransfection solutions were used following manufacturer's suggestions ifavailable (A549, MCF7, SK-LMS-1, 92-1, PC-12). For HMEC-1 and RF/6A(ATCC CRL-1780), AMAXA® HMVEC-L solution together with NUCLEOFECTOR™ IIprogram T-023 were used. For HMC-1, AMAXA® solution V and NUCLEOFECTOR™II program T-030 were used.

After transfection, cells were seeded in 6 well plates using thefollowing media. A549, 92-1, and MCF7 cells were cultured in RPMI(INVITROGEN™, Carlsbad, Calif.) supplemented with 10% fetal bovine serum(FBS) (Hyclone, Logan, Utah), SK-LMS-1 cells were cultured in DMEM(INVITROGEN™, Carlsbad, Calif.), supplemented with 10% FBS (Hyclone,Logan, Utah), PC-12 cells were cultured in F-12K medium (INVITROGEN™,Carlsbad, Calif.), HMC-1 cells were cultured in Iscove's minimum medium(INVITROGEN™) supplemented with 10% FBS and 1.2 mmol/L ofmonothioglycerol (Sigma-Aldrich, St Louis, Mo.). HMEC-1 cells werecultured in EMB-2 (CLONETICS®, San Diego, Calif.) supplemented withEGM-2 MV SingleQuots® (CLONETICS®). PAE cells were cultured in DMEM/F121:1 medium (Invitrogen). RF/6A (ATCC CRL-1780) cells were cultured inRPMI (INVITROGEN™) supplemented with 10% FBS.

After 18 hours transfection efficiency was assessed under a fluorescentmicroscope and when considered appropriate (>40% transfectionefficiency) cells were exposed to 800 μg/ml GENETICIN® (INVITROGEN™).For all cells types, antibiotic resistant clones showed a high range offluorescence intensities including clones (high proportion in some celltypes such as A549) which were negative. In order to enrich for positiveclones and to obtain a more homogeneous population of fluorescent cellsthe cells were sorted by FACS gated on the fluorescent signal of thecells. In some cases, such as the A549 cells, several cycles of cellsorting were used to obtain a population stably transfected and withhomogeneous fluorescence. Once cells were confirmed to be stabletransfectants with homogeneous fluorescence levels map testing andmycoplasma testing were performed.

Example 2 Growth Assay

This example describes exemplary procedures for measuring the growth ofthe cell lines disclosed herein as either monocultures or co-cultures oftwo or more cell lines.

Co-cultures of different cell lines were performed, with each cell lineexpressing a different fluorophore than the other (for example MCF7-RFPexpressing red fluorescent protein and PAE-YFP expressing yellowfluorescent protein). The co-cultures were grown in black, clear bottomCostar 96-well plates (Corning Costar Corp., Cambridge, Mass.). A directrelationship between fluorescence and number of cells in culture wasestablished for all different fluorescent cell lines tested (see forexample FIG. 2).

Different densities of cells were used in different trials. Fluorescenceintensity was obtained using an INFINITE™ M200 (TECAN® Group Ltd.Switzerland) fluorometer. The spectra for the different fluorophoresused overlap at maximum excitation/emission. In order to avoid spectralbleed through and discriminate fluorescence from different cells types,two systems were used. In the first system measurements were taken atsuboptimal emission/excitation wavelengths ensuring no overlap.Precisely gated fluorescence emission and excitation wavelengths allowedthe complete discrimination of fluorescence emitted by different celltypes in a co-culture. As shown in FIG. 3A, when fluorescence emissionis measured in the yellow range, only the cells emitting in the yellowrange (i.e. PAE endothelial cells expressing YFP) show a linearrelationship with the number of cells, while cell emitting in the redrange (i.e. MCF7 cells expressing RFP) do not show such a relationship.As shown in FIG. 3B, when fluorescence emission measurements are done inthe red range, only cells emitting in that range (i.e. MCF7 cellsexpressing RFP) show a linear relationship with cell number.

In the second system measurements obtained at maximumexcitation/emission were corrected through spectral linear unmixingusing a modification of the ImageJ algorithm first implemented by Dr.Joachim Walter (the program is available on the world wide web atrsb.info.nih.gov/ij/plugins/spectral-unmixing.html); based on the workby Timo Zimmermann (Zimmermann, “Spectral imaging and linear unmixing inlight microscopy” Adv Biochem Eng Biotechnol, 95: 245-265, 2005). Thefluorescent cells were also examined under a fluorescent microscopeallowing for morphological analysis and comparison of mono-culturesversus co-cultures, as shown in FIG. 5. Continuous real time readingswere carried out to assay the growth of mono- and co-cultures of PAE andMCF7 cells (see for example FIGS. 4A and 4B).

Example 3 Tubule Formation Assay

This example describes exemplary procedures for measuring the ability ofthe cell lines disclosed herein for tubule formation potential. 50 μl oflow growth factor BME (Basement Membrane Extract, Trevigen, Inc.Gaithersburg, Md.) were laid down in each well and the plate wasincubated for 1 hour at 37° C. The extract gels at 37° C. to form areconstituted basement membrane and stimulates tubule formation byendothelial cells. The major components of the Basement Membrane Extract(BME) include laminin I, collagen IV, entactin, and heparin sulfateproteoglycan. 15,000 PAE-GFP cells were then added on top of the gelledBME and images were acquired with a fluorescent microscope after 3.5-24hours. Cells were imaged on the BD Pathway™ Bioimager. FIG. 7A shows theimpact of increasing concentrations of suramin (an established inhibitorof tubule formation) (from 0 to 26 μM) on tubule formation by PAE greencells (GFP transfected). FIGS. 7B and 7C shows the dose response of PAEgreen cells to suramin in a tubule formation assay. As expected thesuramin inhibited tubule formation and the EC50 calculated (˜26 μM) isin agreement with the value previously published. Tubule formationassays were quantified using the Java based software AngioApplication™.AngioApplication™ has been specifically designed for the quantificationof tubule formation assays. Several morphological parameters wereassessed in images including the total area of the tubules, the totalnumber of tubules, number of nodes, number of branch points, the numberof tubes per node, or node.

AngioApplication™ was validated by comparison of the published EC50value of suramin (˜26 μM) and the EC50 value of suramin as determinedexperimentally using the disclosed fluorescent cell lines in a tubuleformation assay. As shown in FIG. 7A increasing concentrations ofsuramin causes disruption of tubule formation. AngioApplication™ wasused to automatically assess tube length at several concentrations ofsuramin. Traditionally quantification of tube formation was based in themeasurement of complete (long) tubes in the image. AngioApplication™determined that as the concentration of suramin was increased the numberof long tubules was diminished. As shown in FIG. 7B, usingAngioApplication™ the EC50 of suramin was determined to by approximately26 μM, in close agreement with the published EC50 of suramin. Inaddition, because AngioApplication™ calculates the length for all tubesin the image (including incomplete tubes) a more refined analysis wasperformed using the measurement of short (or incomplete) tubules.Interestingly, AngioApplication™ found that the number of small tubesincreased as a function of suramin concentration. This is in agreementwith the fact that Suramin interferes with the mechanism of tubeformation. As shown in FIG. 7C, the EC50 calculated for suramin usingthis additional parameter was approximately 26 μM. This resultdemonstrates that both measurements (short and long tubes) can be usedto evaluate potency of antiangiogenic drugs and potentially otherproangiogenic factors found in patient serum samples.

Example 4 Migration Assay

This example describes exemplary procedures for measuring the ability ofthe cell lines disclosed herein to migrate in response to a chemicalstimulation. In this example migration is up a chemical gradient, from aregion of lower concentration to higher concentration of a chemicalattractant.

Migration assays were performed using the ChemoTx® 96 well cellmigration system (Neuro Probes Inc.) following the manufacturer'srecommendations. Every plate contained an internal negative (absence ofstimulus) and a positive (presence of a known chemotactic substance)control together with wells containing different cell densities whichallow for the construction of standard curves which mathematicallycorrelate number of cells and fluorescence intensity. Different putativechemotactic factors were assayed using this system including plasmaobtained from patients. Fluorescence measurements were obtained using anINFINITE™ M200 fluorescence plate reader to measure the accumulation offluorescent cells in the lower chamber of the ChemoTx® 96 plate that hadmigrated through the membrane to the lower chamber in response tochemoattractant in the lower chamber. Migration can be quantitated andthe relative migration determined by determining the intensity of thefluorescent signal, for example at the emission maxima, of thefluorescent cells in the lower chamber, wherein greater fluoresceintensity in the lower chamber relative to the control indicates thatmigration has occurred. The migration potential of various samples canbe correlated with characteristics of the sample or the subject fromwhich the sample was taken. In several examples, the fluorescent cellsdisclosed herein were tested for migratory potential in the presence ofenhancers/blockers of migration.

Using this migration assay the presence of biologically activechemotactic factors in a sample can be tested. FIG. 8A shows the effectof decreasing human normal serum on the motility of YFP expressing PAEendothelial cells. PEA cells were placed on the upper membrane of theChemoTx® 96 well cell migration plate. The migration of PAE cells to thelower chamber as a function of concentration of human normal serum wasthen determined using an INFINITE™ M200 fluorescence plate reader tomeasure the accumulation of fluorescent cells in the lower chamber. Asexpected, as the serum concentration increases (and therefore thepresence of chemotactic factors increases in the serum) higher levels offluorescence are detected, which are directly related to the migratorycapacity of the cells. This assay was also directly applied to theassessment of the migratory potential of patients' serum samples. FIG. 8shows a study wherein the serum of different patients with or withouttumors and with or without treatments was screened for induced migratorypotential. As expected all samples show higher induced migratorypotential than the negative control (un-stimulated cells). For example,greater or less migratory potential can be correlated with the presenceor absence of tumors or tumor types.

FIGS. 11A and 11B shows the applicability of the endothelial fluorescentcells to assess the angiogenic status of clinical samples. In this casethe migratory potential of the sputum from Idiopatic Pulmonary Fibrosis(IPF) patients was tested to determine the effect of the sputum on themigratory potential of PAE cells expressing fluorescent protein. Thisexample assesses whether angiogenic factors are present in this type ofsample and can be used in combination with the experimental proceduresdescribed in this patent application as a diagnostic/prognostic endpoint. Sputum obtained from the patients was placed in the lower chamberof the ChemoTx® 96 well cell migration system. The migration of PAEcells through the membrane to the lower chamber in response to thesputum was then determined using an INFINITE™ M200 fluorescence platereader to measure the accumulation of fluorescent cells in the lowerchamber. First, a standard curve was generated with one of the samplesfrom an IPF patient demonstrating that it contains chemotactic factorsfor endothelial cells (see FIG. 8A). Subsequently, sputum obtained from13 normal subjects was compared to sputum obtained from 13 IPF patients(see FIG. 8B). As shown in FIG. 8B, sputum from normal subjects does notinduce migration above the phosphate buffered saline (PBS) control.However, samples from IPF patients showed a strong migratory potential.

This assay provides a fast and reliable system to detect the presence ofbiologically active enhancers (for example tumor-derived enhancers), orsuppressors (for example test agents, such as drugs) of cellularmigration in patients' samples.

Example 5 Cell Viability Assay

This example describes exemplary procedures for measuring thecytotoxicity of an agent on the cell lines disclosed herein.

Increasing concentrations of Triton® X were added to fluorescent cellsand after 20 minutes supernatants were collected and transferred to adifferent plate and measured at the appropriate wavelength. As shown inFIG. 9, the relative fluorescence as a function of the log ofconcentration produces a sigmoidal curve correlating the amount ofTriton® X to the percentage of cytotoxicity. Using standard curvefitting software (such as a KaleidaGraph® available from SynergySoftware) the EC50 and other parameters commonly used to assesscytotoxicity can be calculated.

Example 6 Exemplary Screen of Small Molecules as Modulators ofAngiogenesis

This example describes exemplary procedures for screening of test agentsas modulators of angiogenesis. A flow-chart representation of anexemplary implementation of a screening procedure is shown in FIG. 10.As shown in FIG. 10 a primary screen of a library of small molecules isdone using growth and tube formation assays (exemplified above inExamples 2 and 3). This primary screen identifies antiangiogeniccompounds which in some cases are cytotoxic. A counterscreen using thedisclosed cell viability assay (as exemplified in Example 5) isperformed to determine those compounds that are cytotoxic.Antiangiogenic compounds which show little or no cytotoxicity areconsidered putative antiangiogenic candidates and move forward to invivo studies.

In some examples, the growth of a cell line of interest is determined inthe presence of a test agent. In some example, the growth of a mixtureof cell lines of interest is determined in the presence of a test agent.In some examples, this is done in a multiwell format, such as a 96 wellplate or a 384 well plate. For example (as exemplified in FIG. 11), theassays are performed in 96 well format which contains negative controls(column 1), positive controls (column 12) and 80 wells containing thesmall molecules to be tested. A sample containing a mixture of thefluorescent cell lines disclosed herein is provided in the wells of themultiwell plate. Test agents are added to the plate either at a singleconcentration or at graded concentrations, for example from about 1picomolar to about 100 millimolar. The growth of the cells in thepresence of the test agent is determined, for example by determining thefluoresce signal attributable to the fluorescent cell line of interestin the well as compared to a control, for example a control well inwhich no test agent has been added. FIGS. 12A and 12B shows a montage ofmicrographs representing an example of one growth assay plate. As shownin FIG. 12, column 1 contains cells that have not been stimulated, suchthat the cells do not proliferate (the background autofluorescence ofthe cells is measured that way) and column 12 shows growth of theendothelial cells upon exposure to a growth factor cocktail. Test wellswill show different levels of cell growth or growth inhibition, based onthe fluorescence quantified from each well. Wells which contain growthinhibitors (hits are defined using the SASD: sum of the average squaredinside-cluster distances, Gagarin et al. J. Biomol. Screen 11:1-12,2006) are shown in white boxes. Agents that cause a measurable decreasein the growth of the fluorescent cell line of interest are potentialinhibitors of angiogenesis. Potential inhibitors of angiogenesis canthen be tested for there effect on migration, and tubule formation, andfor cytotoxicity using the disclosed assays.

In some examples, the effectiveness of the small molecules to inhibittubule formation is determined. Exemplary methods for determining theeffect of an agent on tube formation is given in Example 4. In a someassays, 50 μl of low growth factor BME is laid down in each well of amultiwell plate (such as a 96 well plate) and the plate is incubated for1 hour at 37° C. Test agents are added to the plate either at a singleconcentration or at graded concentrations, for example from about 1picomolar to about 100 millimolar (in some examples the test agents areadded directly to the BME prior to plating). A cell mixture containingabout 15,000 fluorescent PAE cells is then added on top of the gelledBME. The ability of the test agent to block tubule formation isdetermined, for example by comparing the number of tubes or relatedstructures formed in a sample contacted with a test agent relative to acontrol, such as a sample not contacted with a test agent. In someexamples, this is done by eye, for example by visual inspection of thecells with a fluorescent microscope after 3.5-24 hours. In someexamples, images are acquired with a fluorescent microscope after 3.5-24hours and stored, for example digitally. In some examples, quantitativeevaluation of the effectiveness of the small molecules to block tubeformation is assessed using the AngioApplication™ software.AngioApplication™ can compute multiple parameters which including butare not restricted to: single tube length, single tube area, total tubelength, total tube area, node area, total number of tubes, total numberof nodes, single node branching points, total number of branchingpoints, average node branching points average tube length, average tubearea, average node area, etc. Test agents identified as capable ofinhibiting tubule formation are identified as potential angiogenesisinhibitors.

Potential angiogenesis inhibitors can be screened for cytotoxicity usingthe disclosed cytotoxicity assays, such as exemplified by Example 5.Increasing concentrations of potential angiogenesis inhibitors (such asfrom about 1 picomolar to about 100 millimolar) are added to cellmixtures containing fluorescent cell lines with distinguishable emissionspectra. This method permits the cytotoxicity of a potentialangiogenesis inhibitor on multiple cell lines to be determinedsimultaneously. After about 20 minutes supernatants are collected andtransferred to a different plate and the fluorescence of thesupernatants is measured at the appropriate wavelength corresponding tothe emission spectra of the distinguishable emission spectra of thefluorescent proteins. The measured emission spectra from each of thedistinguishable emission spectra is then used to determine thecytotoxicity of the potential angiogenesis inhibitor on the fluorescentcell lines in the mixture, for example by determining the EC50 of thepotential angiogenesis inhibitor on the individual cell lines in themixture. Potential angiogenesis inhibitors which show no or littlecytotoxicity are considered putative antiangiogenic candidates and canmove forward to in vivo studies.

Example 7 Tumor Stimulated Angiogenesis in 2D Co-Cultures does notCorrelate with Xenograft Angiogenesis

The stably-transfected fluorescent endothelial cells described hereinenable the detection of angiogenic cell activities such as migration andtubule formation. Examples 3 and 4 demonstrate the stimulation ofangiogenic activities in endothelial cultures incubated with variousangiogenic stimuli. This example illustrates that tubule formation isanalogously induced by tumor cells in 2D co-cultures, but that theangiogenic potential of particular tumor cell types in the 2Dco-cultures does not correlate with the angiogenic behavior ofxenografts of the same cell types.

Methods

Unless specified, all methods are as described in the previous examples.

2D Cell Cultures.

Tumor cells were grown in a 96-well plate in the medium previouslydescribed such as RPMI1640, DMEM, or F-12K, in accordance with the celllines used herein, +10% fetal calf serum, to approximately 70%confluence and gently washed three times in PBS. 50 μl gel matrix wereadded and solidified at 37° C. A 100 μl aliquot of endothelial basalmedium-2 (EBM-2) without serum supplementation and containing BEC or LECat between 150,000-300,000 cell/ml were then added on top of thesolidified gel matrix. Cultures were incubated at 37° C. and resultingtube formation determined in 4-6 hours.

Xenografts.

1×10⁶ or 1×10⁷ tumor cells were injected subcutaneously in the hindflank of a nude mouse. A palpable mass is felt under the skin in 1-2weeks having a tumor volume of approximately 50-100 mm³. Mice wererandomized into groups of 10 mice/group having tumor volumes of 50-100mm³, and drug treatment was started at this time. Treatment wascontinued for an additional 2-3 weeks or until tumors reached a maximumvolume of 2000 mm³. For biopsy studies, tumors were excised at a volume<1000 mm³, gross morphology photographs were taken and either sectionedin half for a second gross morphology picture showing internal structureof tumor mass or a core biopsy taken through the entire tumor noduleresulting in a traversing “sausage” tissue sample having peripheral, midsection and center anatomical regions that were ultimately sliced intocross-sections and placed into the 3D drug sensitivity assay (seeExample 8).

Results

Induction of angiogenesis in the tumor microenvironment is a multi-stageprocess, involving multiple factors and cell types (FIG. 20). As shownin Examples 3 and 4, stably-transfected fluorescent endothelial cellscan be used to model angiogenic activities in vitro in response tochemical stimuli provided in culture medium. Tubule formation wasmonitored in Example 3 in 2D cultures that were prepared withendothelial cells layered on top of solidified gel matrix (FIG. 21 topright).

To determine the influence of tumor cells on endothelial tubuleformation, tubule formation was monitored in modified 2D co-cultures ofendothelial cells layered on top of gel matrix that was solidified ontop of a monolayer of a tumor cell line (FIG. 21, top center). Usingthis modified 2D co-culture assay, tubule formation in fluorescent PAEcells was stimulated by several different tumor cell lines and observedafter a six-hour incubation (FIG. 22). Of the cell lines tested, ocularmelanoma 92-1 cells displayed the least tubule inductive potential,while lung carcinoma A549 induced robust tubule formation.

The separate influences of three tumor cell lines (lung carcinoma A549,pheochromocytoma PC-12 (CRL-1721), and ocular melanoma 92-1) on tubuleformation in three endothelial cell lines (PAE, HMEC-1, and LEC-1) wassimilarly tested. As shown in FIG. 23, A549 cells induced robust tubuleformation in all endothelial cells tested. In contrast, 92-1 cells didnot stimulate tubule formation in any of the cells tested.

One goal of the modified 2D co-culture assays was to develop a mimeticof the in vivo effects of a tumor on angiogenesis. To establish thecorrelation between the modified 2D co-culture results and in vivo tumoractivity, tumor xenografts were produced in nude mice using lungcarcinoma A549, pheochromocytoma PC-12, and ocular melanoma 92-1 cells.Resultant tumors were excised and blood vessel formation observed in theperiphery (FIG. 24, top panels) and interior (FIG. 24, bottom panels) ofthe tumors. PC-12 and 92-1 tumors had abundant vasculature both on theperiphery as well as the interior of the tumors. In contrast, the A549tumor displayed moderate vascularization on the periphery of the tumor,but little vasculature in the tumor interior.

These xenograft results strongly diverge from the induction of tubuleformation observed in the modified 2D co-cultures. Thus, the modified 2Dco-cultures cannot serve as a mimetic of in vivo tumor-inducedangiogenesis.

Example 8 Recapitulation of In Vivo Tumor Activities in 3D Co-Cultures

As shown in Example 7, tumor cell-induced endothelial tubule formationin modified 2D co-cultures does not correlate with angiogenesis in acorresponding nude mouse xenograft model. This example describes a 3Dco-culture assay system that accurately recapitulates the in vivoactivity of tumor xenografts. Migration of tumor cells along endothelialtubules, or angiotropism, was also observed in the described 3Dco-culture system. Thus, the 3D co-cultures provide a model to monitorboth angiogenic and metastatic potential of a tumor.

Methods

Unless specified herein, methods were as described in the precedingexamples.

Tumor Spheroids.

Tumor spheroid colonies were prepared according to a modified protocolbased on Hamburger et al. (Science, 197:461-463, 1977). 2% SEAPLAQUE®Agarose (FMC BioProducts) was prepared in deionized water andautoclaved. The agarose was cooled in a 40° C. water bath. 10×RPMI 1640medium (Sigma-Aldrich), FBS, Antibiotic-Antimycotic (a.k.a. Anti-Anti),and sterile deionized water (Invitrogen) were warmed in a 40° C. waterbath. Appropriate volumes of the deionized water, 100×Antibiotic-Antimycotic, FBS, 10× RPMI1640 medium, and 2% agarose weremixed to make the final concentration of 1% agarose with 20% FBS, 2×Antibiotic-Antimycotic, and 1×RPMI1640. 1.5 ml of the mixture was addedto each well of 6-well plate (Corning) and set aside to solidify for 20minutes in the hood. In the meantime, tumor cells at approximately 70%confluence were harvested and counted. Tumor cells were suspended at15,000 cells/ml in 0.2% of agarose, 2× Antibiotic-Antimycotic, 20% FBS,and 1×RPMI1640. 3 ml of the tumor cell suspension was added to the wellwith the first solidified layer in the 6-well plate. This plate was leftin the hood for 10 minutes and then carefully transferred to anincubator with 100% humidity for 20-30 days. The well-formed colonieswere harvested by adding 2 ml of 1×PBS to each well of a 6-well plateand pipetted up and down for sufficient number of times until theagarose was broken into tiny pieces. The colonies were washed threetimes in PBS to get rid of the agarose residue. The colonies weresuspended in sterile PBS with 1% glucose, 0.3 mM EDTA, 0.5% BSA and 1×Antibiotic-Antimycotic. The bright fluorescent colonies were picked upusing an Olympus inverted fluorescent microscopy (Olympus, Japan) for 3Dco-culture.

Xenograft Biopsy.

Xenografts were prepared as in Example 7. Tumor xenografts weredissected when they reached about 1 cm in diameter and put into thesterile 15 ml or 50 ml tubes (Corning) with RPMI1640 medium supplementedwith 10% FBS, 1% Glucose (Sigma), and 4× Antibiotic-Antimycotic(Invitrogen). The tumor xenografts were placed on wet ice and shipped tothe lab within 2-3 hours. The Xenografts were rinsed 3 times in 70%ethanol and then 3 times in PBS. In the hood, the core biopsy wasperformed by biopsy punch (Miltex, Inc.) and was washed out into a 100cm cell culture dish (Corning) by using a pipette to blow the topopening of the biopsy applicator. Using a disposable scalpel (FeatherSafety Razor, Co. Japan), the core biopsy was dissected in three stages.1 mm of both ends of the core biopsy was carefully cut off; they weretransferred into a new 100 cm cell culture dish labeled P (peripheralsection). Next, 1 mm of both ends of the remaining core biopsy wereremoved and discarded. 1 mm of both ends of the core biopsy were cut offand transferred into a new 100 cm cell culture dish labeled M (middlesection). Lastly, 1 mm of both ends of the remaining biopsy tissue wereremoved and discarded. The rest of the core biopsy was transferred intoa new 100 cm cell culture dish labeled C (center section). A drop of PBSwas added to each section P, M, and C to keep them moist. Using thedisposable scalpel, each section was cut into small pieces,approximately 10 pieces per 1 mm section, under the dissectionmicroscope (LeicaMZ125, Leica, Germany). For the 3D co-cultures, eachpiece of the biopsy was transferred to the center of the well of thesecond layer of the gel matrix suspension comprising individualendothelial cells and/or other component cells in a prepared 96-wellplate kept on wet ice. The plate was then placed in the incubator at 37°C. for 45 minutes to allow the gel matrix to solidify. The third layercomprising the liquid medium was then added to the wells for culturing.

3D Co-Cultures.

3D co-cultures were prepared as follows: 50 μl 1% SEAKEM™ LE agarosewere added to individual wells of a 96-well plate and allowed tosolidify at room temperature for 20 minutes. 30 μl GELTREX® gel matrix(Invitrogen) were combined with PAE, LEC or HMEC-1 cells at a celldensity of 560,000 cells/ml and added to each well of the plate (atopthe solidified agarose). Plates were maintained on wet ice (4° C.) toprevent solidification of the gel matrix/cell mixture (the secondlayer). A single tumor cell spheroid colony or single ringlet ofxenograft core biopsy was added to the center of the well and the matrixgel was solidified at 37° C. On top of the tumor/endothelial cell gellayer, 80 μl EBM-2+1% FBS were added to a final concentration ofEMB-2+0.5% FBS in relation to the total volume of the culture. Cultureswere incubated at 37° C. for 5-20 days and endothelial vessel networkformation was observed by confocal laser scanning fluorescencemicroscopy.

Results

Modified 2D co-cultures (Example 7) enabled observation of induction ofendothelial tubule formation by a tumor cell line. However, angiogenesisin the modified 2D co-cultures did not correlate with in vivo tumoractivity in nude mouse xenografts.

In order to more accurately reproduce the tumor microenvironmentillustrated in FIG. 20, 3D co-cultures were developed (FIG. 21, bottom).In the 3D co-cultures, tumor and endothelial cells were mixed togetherin gel matrix (second layer) and layered on top of agarose (first layer)that had been previously solidified in the wells of a 96-cell cultureplate. Culture media (third layer) was provided on top of thetumor/endothelial cell gel layer (second layer).

Employing the 3D co-culture assay, endothelial tubule formation wasobserved in the presence of tumor xenograft tissue (FIG. 25) and singletumor spheroid colonies (FIGS. 26-29). Tubule formation was not observedin co-cultures between endothelial cells and dispersed tumor cells,though endothelial cell migration was observed.

In contrast to the 2D co-cultures, tubule formation in the 3Dco-cultures recapitulated in vivo angiogenesis in the nude micexenografts. Specifically, ocular melanoma 92-1, which produced a highlyvascularized xenograft tumor, induced robust tubule formation after anine-day incubation (FIG. 26) in the described 3D co-culture system.Moreover, lung carcinoma A549 cells, which produced a xenograft tumorthat was only poorly and peripherally vascularized, induced moderateperipheral tubule formation after a twelve-day incubation (FIG. 28) inthe described 3D co-culture system. Similar results were observed forboth tumor cell lines after a twenty-day incubation (FIG. 29). Thus, thedescribed 3D co-cultures provide an in vitro model for angiogenesis thatdirectly correlate with in vivo tumor activity.

Evaluation of highly metastatic human tumor cell lines such aspheochromocytoma or melanoma in the 3D in vitro co-culture model systemalso demonstrated migration of individual cancer cells along vascularhighways (FIGS. 27 and 29). This cellular migration extended far beyondcellular branch projections from the main tumor spheroid seed colony.Vessel-mediated cancer cell migration is known as angiotropism, and hasbeen reported to occur in pathological specimens of humanglioma/glioblastoma and melanoma (Lugassy et al., Am. J. Dermatopath.,24:473-478, 2002; and Lugassy and Barnhill, Adv. Anat. Pathol.,14:195-201, 2007). Thus, in addition to providing an in vitro model fortumor-induced angiogenesis, the 3D co-cultures also provide a model fortumor metastasis.

Example 9 Methods of Testing Anti-Angiogenic Tumor Therapies Using 3DCo-Cultures

The 3D co-cultures described herein provide an in vitro model thatcorrelates with in vivo tumor induction of angiogenesis. With the 3Dco-cultures, it becomes possible to design individualizedanti-angiogenic tumor therapies that are tailored to best inhibitinduction of angiogenesis by a tumor in a subject. This example showsthe testing of anti-angiogenesis treatments using the 3D co-cultureassay of endothelial tubule formation.

Methods

Unless specified, all methods were as described in the previousexamples.

3D Cell Cultures.

3D cultures were prepared as in Example 8, except EBM-2 medium wasprovided on top of the solidified tumor/endothelium/gel layer containingfinal concentrations of 0.5% FBS and 0.1% DMSO, plus angiogenesisinhibitor. Final drug concentrations were as follows: Avastin® at 100μg/ml; Thalidomide at 100 μM; Sunitinib at 6 μg/ml; and Fumagilin at 1μM.

Results

Tumor cell-induced endothelial cell tubule formation in 3D co-culturescorrelated with angiogenesis in tumor xenografts. The 3D co-cultures cantherefore be used to monitor the in vitro angiogenic potential of tumortissue isolated from a subject. In particular, the efficacy of multipleangiogenesis inhibitors in vitro can be tested and monitored with a highdegree of predictive correlation with the in vivo context.

To examine the utility of the 3D co-cultures to test the effects of anangiogenesis inhibitor, 3D co-cultures were prepared combining stablytransfected fluorescent PAE cells and biopsy tissue from peripheral orcentral tissue of a leiomyosarcoma HTB-88 xenograft. The preparedco-cultures were incubated for six days with the FDA-approvedangiogenesis inhibitor Avastin®. As shown in FIG. 30, Avastin® treatmentinhibited both PAE proliferation as well as tubule formation. Moreover,regional differences of biopsy material (periphery, midsection, core) inthe tumor angiogenic potential were observed, with outer geographictumor areas always demonstrating dramatically more vessel induction thaninner tissue regions (FIG. 30, compare top and bottom panels).

Identifying the most efficacious therapy for a particular patient is acrucial goal for successful cancer treatment. To demonstrate the use ofthe 3D co-culture assay for tailoring anti-angiogenesis treatment to aparticular tumor from a particular patient, the effect of severaldifferent angiogenesis inhibitors on tubule formation in 3D co-cultureswas tested. Several FDA approved anti-angiogenic compounds were assessedin this capacity, including Avastin®, Thalidomide, and Sunitinib as wellas the non-FDA approved drug Fumagillin. After a five-day incubation,Avastin® demonstrated the greatest inhibition of proliferation andtubule formation.

Together, these observations validate the use of 3D co-cultures topersonalize anti-angiogenesis cancer therapy and monitor the efficacy ofthe chosen treatment. The existence of tumor heterogeneity was alsoobserved.

Example 10 Testing Anti-Metastatic Tumor Therapies Using 3D Co-Cultures

Example 9 demonstrated that the 3D co-cultures described herein can beused to test the efficacy of a panel of anti-angiogenic tumor therapiesfor given subject. The tumor cell angiotropism observed in FIGS. 27 and29 indicates that tumor metastasis can also be detected using the 3Dco-cultures. This example provides a method for optimizinganti-metastatic therapy to inhibit metastasis in a subject.

3D co-cultures can be established as described herein. For example,co-cultures can be prepared in several wells of a 96-well culture plate.Tumor cells used in the co-culture can be a sample of a tumor biopsyfrom a subject or a spheroid colony derived from a tumor biopsy from asubject. As with the anti-angiogenic compounds tested in Example 9, oneor more anti-metastatic compounds can be combined with culture mediumand applied to some but not all of the established 3D co-cultures. The3D co-cultures are incubated for any length of time sufficient to detectangiotropism, such as nine days, and angiotropism observed under amicroscope. The relative efficacy of an anti-metastatic compound isdetermined by the comparative inhibition of angiotropism in relation,for instance, to the 3D co-culture that did not receive anyanti-metastatic compound. Those compounds with the strongest inhibitoryeffect on angiotropic cell motility (for example, the distance movedfrom core tumor cell colony) are most efficacious.

Example 11 Personalized Anti-Angiogenic or Anti-Metastatic CancerTherapy

This example provides representative methods of selecting and monitoringthe efficacy of an anti-angiogenic or anti-metastatic cancer therapy fora specific subject.

Using the 3D co-cultures described herein, cancer therapy can betailored to a specific subject and the effectiveness of the therapymonitored over time. A tumor biopsy or other cancer cell sample from apatient (the target patient or target subject) can be the source ofcancerous tissue for incorporation into multiple 3D co-cultures. Asdescribed Examples 9 and 10, the 3D co-cultures can be used to selectthe most efficacious anti-angiogenic and/or anti-metastatic compound forthe target patient, from among a panel of anti-angiogenic oranti-metastatic drugs. Additionally, the effects of combining theselected anti-angiogenic and anti-metastatic drugs can also be observed.The selected compound(s) can then be administered to the target patientas part of an anti-cancer therapy regimen.

The continued efficacy of the selected compound(s) can be monitored byobtaining a tumor biopsy from the subject after a given period of time,for example three months, and preparing 3D co-cultures to observe theeffect of the administered treatment on angiogenesis and/or metastasis.If the development drug resistance is observed, a new personalizedtreatment can then identified using the foregoing methods.

In view of the many possible embodiments to which the principles of ourinvention may be applied, it should be recognized that the illustratedembodiment is only a preferred example of the invention and should notbe taken as a limitation on the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

We claim:
 1. A method for monitoring angiogenic or metastatic potentialof tumor cells comprising: preparing a three-dimensional co-culturecomprising: a first layer comprising a neutral polysaccharide polymergel in contact with the bottom of a culture dish; a second layer on topof the first layer, comprising: a solidified gel matrix; endothelialcells dispersed in the solidified gel matrix; and tumor cells comprisingeither a tumor spheroid colony or a sample of a tumor biopsy, suspendedin the solidified gel matrix; and a third layer on top of the secondlayer, comprising culture medium; incubating the three-dimensionalco-culture; and detecting at least one of endothelial cellproliferation, endothelial cell tubule formation or tumor cellangiotropism of the cells in the second layer.
 2. The method of claim 1,wherein the neutral polysaccharide polymer gel comprises agarose.
 3. Themethod of claim 1, wherein the endothelial cells stably andconstitutively express a fluorescent protein.
 4. The method of claim 3,wherein the tumor cells stably and constitutively express a fluorescentprotein with a different emission spectrum from the fluorescent proteinexpressed by the endothelial cells.
 5. The method of claim 3, whereinthe second layer further comprises at least one additional mammaliancell type dispersed in the solidified gel matrix, and wherein the atleast one additional mammalian cell type stably and constitutivelyexpresses a fluorescent protein with a different emission spectrum fromthe fluorescent protein expressed by the endothelial cells.
 6. Themethod of claim 4, wherein the second layer further comprises at leastone additional mammalian cell type dispersed in the solidified gelmatrix, and wherein the at least one additional mammalian cell typestably and constitutively expresses a fluorescent protein with adifferent emission spectrum from either of the fluorescent proteinsexpressed by the endothelial cells or the tumor cells.
 7. The method ofclaim 1, wherein the second layer further comprises at least oneadditional mammalian cell type dispersed in the solidified gel matrix.8. The method of claim 7, wherein the at least one additional mammaliancell type is a cell selected from the group consisting of macrophage,mast cell, fibroblast, adipocyte, and pericyte.
 9. The method of claim1, wherein the first, second, or third layer further comprises at leastone test agent.
 10. The method of claim 9, wherein the test agent is aknown or potential inhibitor of angiogenesis or metastasis.
 11. Themethod of claim 1, wherein the tumor cells are derived from a subjectand the first, second, or third layer further comprises at least onetest agent that has been administered to the subject as part of a cancertreatment.
 12. A method of testing the efficacy of an anti-angiogenic oranti-metastatic cancer treatment for a subject, comprising monitoringangiogenic or metastatic potential of tumor cells by the method of claim1, wherein the tumor cells are derived from the subject and the first,second, or third layer comprises at least one test agent that is acandidate anti-cancer treatment.
 13. A method of selecting apersonalized anti-angiogenic or anti-metastatic treatment for cancer ina subject comprising: preparing multiple three-dimensional co-cultures,each co-culture comprising: a first layer comprising a neutralpolysaccharide polymer gel in contact with the bottom of a culture dish;a second layer on top of the first layer, comprising: a solidified gelmatrix; endothelial cells dispersed in the solidified gel matrix; andtumor cells comprising either a tumor spheroid colony or a sample of atumor biopsy, suspended in the solidified gel matrix; and a third layeron top of the second layer, comprising culture medium, wherein all butone of the co-cultures further comprises at least one test agentcomprising an anti-angiogenic or anti-metastatic compound in the first,second, or third layers; incubating the three-dimensional co-cultures;detecting at least one of endothelial cell proliferation, endothelialcell tubule formation or tumor cell angiotropism of the cells in thesecond layer; and selecting the at least one test agent having thegreatest effect on at least one of endothelial cell proliferation,endothelial cell tubule formation or tumor cell angiotropism incomparison to endothelial cell proliferation, endothelial cell tubuleformation or tumor cell angiotropism in the cells of the co-culturewithout the test agent in the medium.
 14. The method of claim 13,wherein the neutral polysaccharide polymer gel comprises agarose. 15.The method of claim 13, wherein the endothelial cells stably andconstitutively express a fluorescent protein.
 16. The method of claim15, wherein the tumor cells stably and constitutively express afluorescent protein with a different emission spectrum from thefluorescent protein expressed by the endothelial cells.
 17. The methodof claim 15, wherein the second layer further comprises at least oneadditional mammalian cell type dispersed in the solidified gel matrix,and wherein the at least one additional mammalian cell type stably andconstitutively expresses a fluorescent protein with a different emissionspectrum from the fluorescent protein expressed by the endothelialcells.
 18. The method of claim 16, wherein the second layer furthercomprises at least one additional mammalian cell type dispersed in thesolidified gel matrix, and wherein the at least one additional mammaliancell type stably and constitutively expresses a fluorescent protein witha different emission spectrum from the either of the fluorescentproteins expressed by the endothelial cells or the tumor cells.
 19. Themethod of claim 13, wherein the second layer further comprises at leastone additional mammalian cell type dispersed in the solidified gelmatrix.
 20. The method of claim 19, wherein the at least one additionalmammalian cell type is a cell type selected from the group consisting ofmacrophage, mast cell, fibroblast, adipocyte, and pericyte.
 21. Themethod of claim 1, wherein the monoclonal tumor spheroid colony isproduced using a method comprising: preparing a culture in whichmonoclonal tumor spheroid colonies are grown, comprising: a bottom layercomprising about 1% agarose; and a top layer overlaying the bottomlayer, wherein the top layer comprises isolated tumor cells suspended inabout 0.2% of agarose; incubating the culture to grow monoclonalspheroid colonies; harvesting monoclonal tumor spheroid colonies fromthe culture; and resuspending the monoclonal tumor spheroids in abuffered solution.